Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5)

ABSTRACT

This invention provides methods of modifying feeding behavior, including increasing or decreasing food consumption, e.g., in connection with treating obesity, bulimia or anorexia. These methods involve administration of compounds are selective agonists or antagonists or the Y5 receptor. One such compound has the structure:  
                 
In addition, this invention provides an isolated nucleic acid molecule encoding a Y5 receptor, an isolated Y5 receptor protein, vectors comprising an isolated nucleic acid molecule encoding a Y5 receptor, cells comprising such vectors, antibodies directed to the Y5 receptor, nucleic acid probes useful for detecting nucleic acid encoding Y5 receptors, antisense oligonucleotides complementary to any unique sequences of a nucleic acid molecule which encodes a Y5 receptor, and nonhuman transgenic animals which express DNA a normal or a mutant Y5 receptor.

This application is a continuation-in-part of U.S. Ser. No. 08/349,025,filed Dec. 2, 1994, the contents of which are hereby incorporated byreference into the subject application.

BACKGROUND OF THE INVENTION

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the sequence listing and the claims.

Neuropeptide Y (NPY) is a member of the pancreatic polypeptide familywith widespread distribution throughout the mammalian nervous system.NPY and its relatives (peptide YY or PYY, and pancreatic polypeptide orPP) elicit a broad range of physiological effects through activation ofat least five G protein-coupled receptor subtypes known as Y1, Y2, Y3,Y4 (or PP), and the “atypical Y1”. The role of NPY as the most powerfulstimulant of feeding behavior yet described is thought to occurprimarily through activation of the hypothalamic “atypical Y1” receptor.This receptor is unique in that its classification was based solely onfeeding behavior data, rather than radioligand binding data, unlike theY1, Y2, Y3, and Y4 (or PP) receptors, each of which were describedpreviously in both radioligand binding and functional assays. Applicantsnow report the use of a ¹²⁵I-PYY-based expression cloning technique toisolate a rat hypothalamic cDNA encoding an “atypical Y1” receptorreferred to herein as the Y5 subtype. Applicants also report theisolation and characterization of a Y5 homolog from human hippocampus.Protein sequence analysis reveals that the Y5 receptor belongs to the Gprotein-coupled receptor superfamily. Both the human and rat homologdisplay≦42% identity in transmembrane domains with the previously cloned“Y-type” receptors. Rat brain localization studies using in situhybridization techniques verified the existence of Y5 receptor mRNA inrat hypothalamus. Pharmacological evaluation revealed the followingsimilarities between the Y5 and the “atypical Y1” receptor. 1) Peptidesbound to the Y5 receptor with a rank order of potency identical to thatdescribed for the feeding response: NPY≧NPY₂₋₃₆=PYY=[Leu³¹,Pro³⁴]NPY>>NPY₁₃₋₃₆. 2) The Y5 receptor was negatively coupled to cAMPaccumulation, as had been proposed for the “atypical Y1” receptor. 3)Peptides activated the Y5 receptor with a rank order of potencyidentical to that described for the feeding response. 4) The reportedfeeding “modulator” [D-Trp³²]NPY bound selectively to the Y5 receptorand subsequently activated the receptor. 5) Both the Y5 and the“atypical Y1” receptors were sensitive to deletions or modifications inthe midregion of NPY and related peptide ligands. These data support theidentity of the Y5 receptor as the previously described “atypical Y1”,and furthermore indicate a role for the Y5 receptor as a potentialtarget in the treatment of obesity, metabolism, and appetite disorders.

The peptide neurotransmitter neuropeptide Y (NPY) is a 36 amino acidmember of the pancreatic polypeptide family with widespread distributionthroughout the mammalian nervous system. NPY is considered to be themost powerful stimulant of feeding behavior yet described (Clark et al.,1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Directinjection into the hypothalamus of satiated rats, for example, canincrease food intake up to 10-fold over a 4-hour period (Stanley et al.,1992). The role of NPY in normal and abnormal eating behavior, and theability to interfere with NPY-dependent pathways as a means to appetiteand weight control, are areas of great interest in pharmacological andpharmaceutical research (Sahu and Kalra, 1993; Dryden et al., 1994). Anycredible means of studying or controlling NPY-dependent feedingbehavior, however, must necessarily be highly specific as NPY can actthrough at least 5 pharmacologically defined receptor subtypes to elicita wide variety of physiological functions (Dumont et al., 1992). It istherefore vital that knowledge of the molecular biology and structuraldiversity of the individual receptor subtypes be understood as part of arational drug design approach to develop subtype selective compounds. Abrief review of NPY receptor pharmacology is summarized below and alsoin Table 1.

Table 1: Pharmacologically Defined Receptors for NPY and RelatedPancreatic Polypeptides.

Rank orders of affinity for key peptides (NPY, PYY, PP, [Leu³,Pro³⁴]NPY, NPY₂₃₆, and NPY₁₃₋₃₆) are based on previously reportedbinding and functional data (Schwartz et al., 1990; Wahlestedt et al.,1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). Data for the Y2receptor were disclosed in pending U.S. patent application Ser. No.08/192,288 filed on Feb. 3, 1994, currently pending, the foregoingcontents of which are hereby incorporated by reference. Data for the Y4receptor were disclosed in pending U.S. patent application Ser. No.08/176,412 filed on Dec. 28, 1993, the contents of which are herebyincorporated by reference. Missing peptides in the series reflect a lackof published information. TABLE 1 Affinity (pK_(i) or pEC₅₀) Receptor 11to 10 10 to 9 9 to 8 8 to 7 7 to 6 <6 Y1 NPY NPY₂₋₃₆ NPY₁₃₋₃₆ PP PYY[Leu³¹, Pro³⁴] NPY Y2 PYY NPY₁₃₋₃₆ [Leu³¹, NPY Pro³⁴]N NPY₂₋₃₆ PY PP Y3NPY [Pro³⁴] NPY₁₃₋₃₆PP PYY NPY Y4 PP PYY NPY NPY₁₃₋₃₆ [Leu³¹, Pro³⁴]NPY₂₋₃₆ NPY atypical PYY NPY₁₃₋₃₆ Y1 NPY (feeding) NPY₂₋₃₆ [Leu³¹,Pro³⁴] NPYNPY Receptor Pharmacology

NPY receptor pharmacology has historically been based onstructure/activity relationships within the pancreatic polypeptidefamily. The entire family includes the namesake pancreatic polypeptide(PP), synthesized primarily by endocrine cells in the pancreas; peptideYY (PYY), synthesized primarily by endocrine cells in the gut; and NPY,synthesized primarily in neurons (Michel, 1991; Dumont et al., 1992;Wahlestedt and Reis, 1993). All pancreatic polypeptide family membersshare a compact structure involving a “PP-fold” and a conservedC-terminal hexapeptide ending in Tyr³⁶ (or Y³⁶ in the single lettercode). The striking conservation of Y³⁶ has prompted the reference tothe pancreatic polypeptides' receptors as “Y-type” receptors (Wahlestedtet al., 1987), all of which are proposed to function as seventransmembrane-spanning G protein-coupled receptors (Dumont et al.,1992).

The Y1 receptor recognizes NPY≧PYY>>PP (Grundemar et al., 1992). Thereceptor requires both the N- and the C-terminal regions of the peptidesfor optimal recognition. Exchange of Gln³⁴ in NPY or PYY with theanalogous residue from PP (Pro³⁴), however, is well-tolerated. The Y1receptor has been cloned from a variety of species including human, ratand mouse (Larhammar et al, 1992; Herzog et al, 1992; Eva et al, 1990;Eva et al, 1992). The Y2 receptor recognizes PYY˜NPY>>PP and isrelatively tolerant of N-terminal deletion (Grundemar et al., 1992). Thereceptor has a strict requirement for structure in the C-terminus(Arg³³-Gln³⁴-Arg³⁵-Tyr³⁶-NH₂) exchange of Gln³⁴ with Pro³⁴, as in PP, isnot well tolerated. The Y2 receptor has recently been cloned (disclosedin U.S. patent application Ser. No. 08/192,288, filed Feb. 3, 1994). TheY3 receptor is characterized by a strong preference for NPY over PYY andPP (Wahlestedt et al., 1991). [Pro³⁴]NPY is reasonably well toleratedeven though PP, which also contains Pro³⁴, does not bind well to the Y3receptor. The Y3 receptor (Y3) has not yet been cloned. The Y4 receptor(disclosed in U.S. patent application Ser. No. 08/176,412, filed Dec.28, 1993) binds PP>PYY>NPY. Like the Y1, the Y4 requires both the N- andthe C-terminal regions of the peptides for optimal recognition (U.S.Ser. No. 08/176,412). The “atypical Y1” or “feeding” receptor wasdefined exclusively by injection of several pancreatic polypeptideanalogs into the paraventricular nucleus of the rat hypothalamus whichstimulated feeding behavior with the following rank order:NPY₂₋₃₆≧NPY˜PYY˜[Leu³¹, Pro³⁴]NPY>NPY₁₃₋₃₆ (Kalra et al., 1991; Stanleyet al., 1992). The profile is similar to that of a Y1-like receptorexcept for the anomalous ability of NPY₂₋₃₆ to stimulate food intakewith potency equivalent or better than that of NPY. A subsequent reportin J. Med. Chem. by Balasubramaniam et al. (1994) showed that feedingcan be regulated by [D-Trp³²]NPY. While this peptide was presented as anNPY antagonist, the published data at least in part support astimulatory effect of [D-Trp³²]NPY on feeding. [D-Trp³²]NPY therebyrepresents another diagnostic tool for receptor identification. Incontrast to other NPY receptor subtypes, the “feeding” receptor hasnever been characterized for peptide binding affinity in radioligandbinding assays and the fact that a single receptor could be responsiblefor the feeding response has been impossible to validate in the absenceof an isolated receptor protein; the possibility exists, for example,that the feeding response could be a composite profile of Y1 and Y2subtypes.

Applicants now report the isolation by expression cloning of a novelY-type receptor from a rat hypothalamic cDNA library, along with itspharmacological characterization, in situ localization, and humanhomologues. The data provided link this newly-cloned receptor subtype,from now on referred to as the Y5 subtype, to the “atypical Y1” feedingresponse. This discovery therefore provides a novel approach, throughthe use of heterologous expression systems, to develop a subtypeselective antagonist for obesity and other indications.

Applicants further report the isolation of a canine Y5 S receptor.

In addition, applicants report the discovery of chemical compounds whichbind selectively to the Y5 receptor of the present invention and whichact as antagonists of the Y5 receptor.

The treatment of disorders or diseases associated with the inhibition ofthe Y5 receptor subtype, especially diseases caused by eating disorderslike obesity, bulimia nervosa, diabetes, dislipidimia, may be effectedby administration of compounds which bind selectively to the Y5 receptorand inhibit the activation of the Y5 receptor. Furthermore, any diseasestates in which the Y5 receptor subtype is involved, for example, memoryloss, epileptic seizures, migraine, sleep disturbance, and pain, mayalso be treated using compounds which bind selectively to the Y5receptor.

SUMMARY OF THE INVENTION

This invention provider a method of modifying feeding behavior of asubject which comprises administering to the subject an amount of acompound which is a Y5 receptor agonist or antagonist effective toincrease or decrease consumption of food by the subject so as to therebymodify feeding behavior of the subject.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of anon-peptidyl compound which is a Y5 receptor antagonist effective toinhibit the activity of the subject's Y5 recetpor, wherein the bindingof the compound to the human receptor is characterized by a K_(i) lessthan 100 nanomolar when measured in the presence of ¹²⁵I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor antagonist effective to inhibitthe activity of the subject's Y5 receptor, wherein the compound'sbinding to the human Y5 receptor is characterized by a K_(i) less than10 nanomolar when measured in the presence of ¹²⁵I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of anon-peptidyl compound which is a Y5 receptor agonist effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 100 nanomolar when measured in the presence of ¹²⁵I-PYY;and (b) the binding of the compound to any other human Y-type receptoris characterized by a K_(i) greater than 1000 nanomolar when measured inthe presence of ¹²⁵I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of anon-peptidyl compound which is a Y5 receptor agonist effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 1 nanomolar when measured in the presence of ¹²⁵I-PYY;and (b) the compound's binding to any other human Y-type receptor ischaracterized by a K_(i) greater than 100 nanomolar when measured in thepresence of ¹²⁵I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor agonist effective to increasethe activity of the subject's Y5 receptor, wherein (a) the binding ofthe compound to the human Y5 receptor is characterized by a K_(i) lessthan 1 nanomolar when measured in the presence of ¹²⁵I-PYY; and (b) thebinding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 25 nanomolar when measured in thepresence of ¹²⁵I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor agonist effective to increasethe activity of the subject's Y5 receptor, wherein (a) the binding ofthe compound to the human Y5 S receptor is characterized by a K_(i) lessthan 0.1 nanomolar when measured in the presence of ¹²⁵I-PYY; and (b)the binding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 1 nanomolar when measured in thepresence of ¹²⁵I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor agonist effective to increasethe activity of the subject's Y5 receptor, wherein (a) the binding ofthe compound to the human Y5 receptor is characterized by a K_(i) lessthan 0.01 nanomolar when measured in the presence of ¹²⁵I-PYY; and (b)the binding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 1 nanomolar when measured in thepresence of ¹²⁵I-PYY.

This invention provides an isolated nucleic acid encoding a Y5 receptor.This invention also provides an isolated Y5 receptor protein. Thisinvention provides a vector comprising the above-described nucleic acid.

This invention provides a plasmid which comprises the regulatoryelements necessary for expression of DNA in a mammalian cell operativelylinked to the DNA encoding the human Y5 receptor as to permit expressionthereof designated pcEXV-hY5 (ATCC Accession No. 75943).

This invention provides a plasmid which comprises the regulatoryelements necessary for expression of DNA in a mammalian cell operativelylinked to the DNA encoding the rat Y5 receptor as to permit expressionthereof designated pcEXV-rY5 (ATCC Accession No. 75944).

This invention provides a mammalian cell comprising the above-describedplasmid or vector.

This invention provides a nucleic acid probe comprising a nucleic acidof at least 15 nucleotides capable of specifically hybridizing with aunique sequence included within the sequence of a nucleic acid encodinga Y5 receptor.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to mRNA encoding a Y5 receptor so asto prevent translation of the mRNA.

This invention provides an antibody directed to a Y5 receptor.

This invention provides a pharmaceutical composition comprising anamount of the oligonucleotide effective to reduce activity of a human Y5receptor by passing through a cell membrane and binding specificallywith mRNA encoding a human Y5 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane.

This invention provides a pharmaceutical composition comprising anamount of an antagonist effective to reduce the activity of a human Y5receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising anamount of an agonist effective to increase activity of a Y5 receptor anda pharmaceutically acceptable carrier.

This invention provides the above-described pharmaceutical compositionwhich comprises an amount of the antibody effective to block binding ofa ligand to the Y5 receptor and a pharmaceutically acceptable carrier.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human Y5 receptor.

This invention also provides a method for determining whether a ligandcan specifically bind to a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding the Y5 receptor with theligand under conditions permitting binding of ligands to such receptor,detecting the presence of any such ligand specifically bound to the Y5receptor, and thereby determining whether the ligand specifically bindsto the Y5 receptor.

This invention provides a method for determining whether a ligand canspecifically bind to a Y5 receptor which comprises preparing a cellextract from cells transfected with and expressing DNA encoding the Y5receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand under conditionspermitting binding of ligands to such receptor, detecting the presenceof the ligand specifically bound to the Y5 receptor, and therebydetermining whether the ligand specifically binds to the Y5 receptor.

This invention provides a method for determining whether a ligand is aY5 receptor agonist which comprises contacting a cell transfected withand expressing nucleic acid encoding a human Y5 receptor with the ligandunder conditions permitting activation of the Y5 receptor, detecting anincrease in Y5 receptor activity, and thereby determining whether theligand is a human Y5 receptor agonist.

This invention provides a method for determining whether a ligand is aY5 receptor antagonist which comprises contacting a cell transfectedwith and expressing DNA encoding a Y5 receptor with the ligand in thepresence of a known Y5 receptor agonist, such as PYY or NPY, underconditions permitting the activation of the Y5 receptor, detecting adecrease in Y5 receptor activity, and thereby determining whether theligand is a Y5 receptor antagonist.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a Y5 receptor to identify a compoundwhich specifically binds to the Y5 receptor, which comprises (a)contacting a cell transfected with and expressing DNA encoding the Y5receptor with a compound known to bind specifically to the Y5 receptor;(b) contacting the preparation of step (a) with the plurality ofcompounds not known to bind specifically to the Y5 receptor, underconditions permitting binding of compounds known to bind the Y5receptor; (c) determining whether the binding of the compound known tobind to the Y5 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to the Y5receptor of each compound included in the plurality of compounds, so asto thereby identify the compound which specifically binds to the Y5receptor. This invention provides a method of screening a plurality ofchemical compounds not known to bind to a Y5 receptor to identify acompound which specifically binds to the Y5 receptor, which comprises(a) preparing a cell extract from cells transfected with and expressingDNA encoding the Y5 receptor, isolating a membrane fraction from thecell extract, contacting the membrane fraction with a compoumd known tobind specifically to the Y5 receptor; (b) contacting preparation of step(a) with the plurality of compounds not known to bind specifically tothe Y5 receptor, under conditions permitting binding of compounds knownto bind the Y5 receptor; (c) determining whether: the binding of thecompound known to bind to the Y5 receptor is reduced in the presence ofthe compounds, relative to the binding of the compound in the absence ofthe plurality of compounds; and if so (d) separately determining thebinding to the Y5 receptor of each compound included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the Y5 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a Y5 receptor to identify a compoundwhich activates the Y5 receptor which comprises (a) contacting a celltransfected with and expressing the Y5 receptor with the plurality ofcompounds not known to bind specifically to the Y5 receptor, underconditions permitting activation of the Y5 receptor; (b) determiningwhether the activity of the Y5 receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the Y5 receptor is increased by each compound included inthe plurality of compounds, so as to thereby identify the compound whichactivates the Y5 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a Y5 receptor to identify a compoundwhich activates the Y5 receptor which comprises (a) preparing a cellextract from cells transfected with and expressing DNA encoding the Y5receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the plurality of compounds notknown to bind specifically to the Y5 receptor, under conditionspermitting activation of the Y5 receptor; (b) determining whether theactivity of the Y5 receptor is increased in the presence of thecompounds; and if so (c) separately determining whether the activationof the Y5 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the Y5 receptor. This invention provides a method of screeninga plurality of chemical compounds not known to inhibit the activation ofa Y5 receptor to identify a compound which inhibits the activation ofthe Y5 receptor, which comprises (a) contacting a cell transfected withand expressing the Y5 receptor with the plurality of compounds in thepresence of a known Y5 receptor agonist, under conditions permittingactivation of the Y5 receptor; (b) determining whether the activation ofthe Y5 receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the Y5 receptor in the absenceof the plurality of compounds; and if so (c) separately determining theinhibition of activation of the Y5 receptor for each compound includedin the plurality of compounds, so as to thereby identify the compoundwhich inhibits the activation of the Y5 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a Y5 receptor toidentify a compound which inhibits the activation of the Y5 receptor,which comprises (a) preparing a cell extract from cells transfected withand expressing DNA encoding the Y5 receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe plurality of compounds in the presence of a known Y5 receptoragonist, under conditions permitting activation of the Y5 receptor; (b)determining whether the activation of the Y5 receptor is reduced in thepresence of the plurality of compounds, relative to the activation ofthe Y5 receptor in the absence of the plurality of compounds; and if so(c) separately determining the inhibition of activation of the Y5receptor for each compound included in the plurality of compounds, so asto thereby identify the compound which inhibits the activation of the Y5receptor.

This invention provides a method of screening drugs to identify drugswhich specifically bind to a Y5 receptor on the surface of a cell whichcomprises contacting a cell transfected with and expressing DNA encodinga Y5 receptor with a plurality of drugs under conditions permittingbinding of drugs to the Y5 receptor, determining those drugs whichspecifically bind to the transfected cell, and thereby identifying drugswhich specifically bind to the Y5 receptor.

This invention provides a method of screening drugs to identify drugswhich act as agonists of a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding a Y5 receptor with aplurality of drugs under conditions permitting the activation of afunctional Y5 receptor response, determining those drugs which activatesuch receptor in the cell, and thereby identify drugs which act as Y5receptor agonists.

This invention provides a method of screening drugs to identify drugswhich act as Y5 receptor antagonists which comprises contacting cellstransfected with and expressing DNA encoding a Y5 receptor with aplurality of drugs in the presence of a known Y5 receptor agonist, suchas PYY or NPY, under conditions permitting the activation of afunctional Y5 receptor response, determining those drugs which inhibitthe activation of the receptor in the mammalian cell, and therebyidentifying drugs which act as Y5 receptor antagonists.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of a Y5receptor which comprises administering to a subject an effective amountof Y5 receptor antagonist.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a Y5 receptorwhich comprises administering to a subject an effective amount of a Y5receptor agonist.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific human Y5 receptorallele which comprises: a. obtaining DNA of subjects suffering from thedisorder; performing a restriction digest of the DNA with a panel ofrestriction enzymes; c. electrophoretic-ally separating the resultingDNA fragments on a sizing gel; d. contacting the resulting gel with anucleic acid probe capable of specifically hybridizing to DNA encoding ahuman Y5 receptor and labelled with a detectable marker; e. detectinglabelled bands which have hybridized to the DNA encoding a human Y5receptor labelled with a detectable marker to create a unique bandpattern specific to the DNA of subjects suffering from the disorder; f.preparing DNA obtained for diagnosis by steps a-e; and g. comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step e and the DNA obtained for diagnosis from step f todetermine whether the patterns are the same or different and to diagnosethereby predisposition to the disorder if the patterns are the same.

This invention provides a method of preparing the isolated Y5 receptorwhich comprises: a. inserting nucleic acid encoding Y5 receptor in asuitable vector which comprises the regulatory elements necessary ofrexpression of the nucleic acid operatively linked to the nucleic acidencoding a Y5 receptor; b. inserting the resulting vector in a suitablehost cell so as to obtain a cell which produces the Y5 receptor; c.recovering the receptor produced by the resulting cell; and d. purifyingthe receptor so recovered.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Competitive displacement of ¹²⁵I-PYY on membranes from rathypothalamus. Membranes were incubated with 125I-PYY and increasingconcentrations of peptide competitors. IC₅₀ values corresponding to 50%displacement were determined by nonlinear regression analysis. Data arerepresentative of at least two independent experiments. IC₅₀ values forthese compounds are listed separately in Table 2.

FIG. 2 Competitive displacement of ¹²⁵I-PYY336 on membranes from rathypothalamus. Membranes were incubated with ¹²⁵I-PYY₃₋₃₆ and increasingconcentrations of peptide competitors. IC₅₀ values corresponding to 50%displacement were determined by nonlinear regression analysis. Data arerepresentative of at least two independent experiments. IC₅₀ values forthese compounds are listed separately in Table 2.

FIG. 3 Nucleotide sequence of the rat hypothalamic Y5 cDNA clone (Seq.I.D. No 1). Initiation and stop codons are underlined. Only partial 5′and 3′ untranslated sequences are shown.

FIG. 4 Corresponding amino acid sequence of the rat hypothalamic Y5 cDNAclone (Seq. I.D. No. 2).

FIG. 5 Nucleotide sequence of the human hippocampal Y5 cDNA clone (Seq.I.D. No. 3). Initiation and stop codons are underlined. Only partial 5′and 3′ untranslated sequences are shown.

FIG. 6 Corresponding amino acid sequence of the human hippocampal Y5cDNA clone (Seq. I.D. No. 4).

FIG. 7A-E. Comparison of coding nucleotide sequences between rathypothalamic Y5 (top row) and human hippocampal Y5 (bottom row) cDNAclones (84.1% nucleotide identity). F-G. Comparison of deduced aminoacid sequences between rat hypothalamic Y5 (top row) and humanhippocampal Y5 (bottom row) cDNA clones (87.2% overall and 98.8%transmembrane domain identities).

FIG. 8 Comparison of the human Y5 receptor deduced amino acid sequencewith those of the human Y1, Y2, Y4 sequences. Solid bars, the sevenputative membrane-spanning domains (TM I-VII). Shading, identitiesbetween receptor sequences.

FIG. 9 Equilibrium binding of ¹²⁵I-PYY to membranes from COS-7 cellstransiently expressing rat Y5 receptors. Membranes were incubated with¹²⁵I-PYY for the times indicated, in the presence or absence of 300 nMhuman NPY. Specific binding, B, was plotted against time, t, to obtainthe maximum number of equilibrium binding sites, B_(max), and observedassociation rate, K_(obs), according to the equation,B=B_(max)*(1−e^(−(kobs*t))). Binding is shown as the percentage of totalequilibrium binding, B_(max), determined by nonlinear regressionanalysis. Each point represents a triplicate determination.

FIG. 10 Saturable equilibrium binding of ¹²⁵I-PYY to membranes fromCOS-7 cells transiently expressing rat Y5 receptors. Membranes wereincubated with ¹²⁵I-PYY ranging in concentration from 0.4 pM to 2.7 nM,in the presence or absence of 300 nM human NPY. Specific binding, B, wasplotted against the free ¹²⁵I-PYY concentration, [L], to obtain themaximum number of saturable binding sites, B_(max), and the ¹²⁵I-PYYequilibrium dissociation constant, K_(d), according to the bindingisotherm, B=B_(max)[L]/([L]+K_(d)). Specific binding is shown. Data arerepresentative of three independent experiments, with each pointmeasured in triplicate.

FIG. 11 Competitive displacement of ¹²⁵I-PYY from COS-7 cellstransiently expressing rat Y5 receptors. Membranes were incubated with¹²⁵I-PYY and increasing concentrations of peptide competitors. IC₅₀values corresponding to 50% displacement were determined by nonlinearregression analysis and converted to K_(i) values according to theequation, K_(i)=IC₅₀/(1+[L]/K _(d)), where [L] is the ¹²⁵I-PYYconcentration and K_(d) is the equilibrium dissociation constant of¹²⁵I-PYY. Data are representative of at least two independentexperiments. Rank orders of affinity for these and other compounds arelisted separately in Table 4.

FIG. 12 Inhibition of forskolin-stimulated cAMP accumulation in intact293 cells stably expressing rat Y5 receptors. Functional data werederived from radioimmunoassay of cAMP in 293 cells stimulated with 10 μMforskolin over a 5 minute period. Rat/human NPY was tested for agonistactivity at concentrations ranging from 0.03 pM to 0.3 μM over the sameperiod. The EC₅₀ value corresponding to 50% maximal activity wasdetermined by nonlinear regression analysis. The data shown arerepresentative of three independent experiments.

FIG. 13 Schematic diagrams of coronal sections through the rat brain,illustrating the distribution of NPY Y5 receptor mRNA, as visualizedmicroscopically in sections dipped in liquid emulsion. The sections arearranged from rostral (A) to caudal (H). Differences in silver graindensity over individual neurons in a given area are indicated by thehatching gradient. The full definitions for the abbreviations are asfollows:

-   -   Aco=anterior cortical amygdaloid nucleus;    -   AD=anterodorsal thalamic nucleus;    -   APT=anterior pretectal nucleus;    -   Arc=arcuate hypothalamic nucleus;    -   BLA=basolateral amygdaloid nucleus anterior;    -   CA3=field CA3 of Ammon's horn, hippocampus;    -   CeA=central amygdaloid nucleus;    -   Cg=cingulate cortex;    -   CL=centrolateral thalamic nucleus;    -   CM=central medial thalamic nucleus    -   DG=dentate gyrus, hippocampus;    -   DMH=dorsomedial hypothalamic nucleus;    -   DR=dorsal raphe;    -   GiA=gigantocellular reticular nucleus, alpha;    -   HDB=nucleus horizontal limb diagonal band;    -   InG=intermediate gray layer superior colliculus;    -   LC=locus coeruleus;    -   LH=lateral hypothalamic area;    -   MePV=medial amygdaloid nucleus, posteroventral;    -   MVe=medial vestibular nucleus;    -   MHb=medial habenular nucleus;    -   MPN=medial preoptic nucleus;    -   PAG=periaqueductal gray;    -   PaS=parasubiculum;    -   PC=paracentral thalamic nucleus;    -   PCRtA=parvocellular reticular nucleus, alpha;    -   Pe=periventricular hypothalamic nucleus;    -   PrS=presubiculum;    -   PN=pontine nuclei;    -   PVH=paraventricular hypothalamic nucleus;    -   PVHmp=paraventricular hypothalamic nucleus, medial parvicellular        part    -   PVT=paraventricular thalamic nucleus;    -   Re=reunions thalamic nucleus;    -   RLi=rostral linear nucleus raphe;    -   RSG=retrosplenial cortex;    -   SCN=suprachiasmatic nucleus;    -   SNc=substantia nigra, pars compacta; and    -   SON=supraoptic nucleus.

FIG. 14 Partial Nucleotide sequence of the canine Y5 cDNA clonebeginning immediately upstream of TM III to the stop codon (underlined).(Seq. I.D. No 5). Only partial untranslated sequences are shown.

FIG. 15 Corresponding amino acid sequence of the canine Y5 cDNA clone(Seq. I.D. No. 6).

FIG. 16 A. Northern blot analysis of various rat tissues. B. Northernblot analysis of various human brain areas: amygdala, caudate nucleus,corpus callosum, hippocampus, whole brain, substantia nigra, subthalamicnucleus, and thalamus. C. Northern blot analysis of various additionalhuman brain areas: cerebellum, cerebral cortex, medulla, spinal cord,occipital lobe, frontal lobe, temporal lobe, and putamen. Hybridizationwas done under conditions of high stringency, as described inExperimental Details.

FIG. 17 Southern blot analysis of human(A) or rat(B) genomic DNAencoding the Y5 receptor subtype. Hybridization was done underconditions of high stringency, as described in Experimental Details.

FIG. 18 Time course for equilibrium binding of ¹²⁵I-Leu³, Pro³⁴-PYY tothe rat Y5 receptor. Membranes were incubated with 0.08 nM radioligandat room temperature for the length of time indicated in binding buffercontaining either 10 mM Na+ or 138 mM Na+.

FIG. 19 Guanine Nucleotide Modulation of Y5 Peptide Binding. Human orrat Y5 receptors transiently expressed in COS-7 cell membranes, or humanY5 receptors stably expressed in LM(tk−) cell membranes, were incubatedwith 0.08 nM ¹²⁵I-PYY and increasing concentrations of Gpp(NH)p asindicated under standard binding assay conditions. Radioligand bindingis reported as cpm, efficiency=0.8. For the human Y5 in LM(tk−) (0.007mg membrane protein/sample), the maximum Δ cpm=−2343. Given a specificactivity of 2200 Ci/mmol, the change in radioligand binding is thereforecalculated to be −0.6 fmol/0.007 mg protein=−85 fmol/mg membraneprotein.

FIG. 20 NPY-Dependent Inhibition of Forskolin Stimulated cAMPAccumulation by Cloned Y5 Receptors. Intact cells stably transfectedwith human or rat Y5 receptors were incubated with forskolin plus arange of human NPY concentrations as indicated. A representativeexperiment is shown for each receptor system (n≧2).

FIG. 21 Calcium Mobilization: Fura-2 Assay. Cloned human Y-typereceptors in the host cells indicated were screened for intracellularcalcium mobilization in response to NPY and related peptides.Representative calcium transients are shown for each receptor system.

-   -   A. Human Y1 receptor    -   B. Human Y2 receptor    -   C. Human Y4 receptor    -   D. Human Y5 receptor

FIG. 22 Structures of Y5-selective compounds. The structures of thecompounds evaluated at the human Y-type receptors are given.

Detailed Description of the-Invention

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases: C = cytosine A = adenine T =thymine G = guanine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the receptors of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which is decreases the activity of any of thereceptors of the subject invention.

The activity of a G-protein coupled receptor such as a Y5 receptor maybe measured using any of a variety of appropriate functional assays inwhich activation of the receptor in question results in an observablechange in the level of some second messenger system, including but notlimited to adenylate cyclase, calcium mobilization, inositolphospholipid hydrolysis or guanylyl cyclase.

This invention provides a method of modifying feeding behavior of asubject which comprises administering to the subject an amount of acompound which is a Y5 receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject. In one embodiment, thecompound is a Y5 receptor antagonist and the amount is effective todecrease the consumption of food by the subject. In a furtherembodiment, the compound is administered in combination with food. Inanother embodiment the compound is a Y5 receptor agonist and the amountis effective to increase the consumption of food by the subject. In afurther embodiment the compound is administered in combination withfood. The subject may be a vertebrate, a mammal, a human or a caninesubject.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of anon-peptidyl compound which is a Y5 receptor antagonist effective toinhibit the activity of the subject's Y5 receptor, wherein the bindingof the compound to the human Y5 receptor is characterized by a K_(i)less than 100 nanomolar when measured in the presence of ¹²⁵I-PYY. Inone embodiment the compound has a K_(i) less than 50 nanomolar. Inanother embodiment, the compound has a K_(i) less than 10 nanomolar. Ina further embodiment, the binding of the compound to any other humanY-type receptor is characterized by a K_(i) greater than 10 nanomolarwhen measured in the presence of ¹²⁵I-PYY. In another embodiment, thebinding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 50 nanomolar. In anotherembodiment, the binding of the compound is characterized by a K_(i)greater than 100 nanomolar. In one embodiment, the compound binds to thehuman Y5 receptor with an affinity greater than ten-fold higher than theaffinity with which the compound binds to any other human Y-typereceptor. In a further embodiment the compound binds to the human Y5receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to each of the human Y1, human Y2 andhuman Y4 receptors. The feeding disorder may be obesity or bulimia. Thesubject may be a-vertebrate, a mammal, a human or a canine subject.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor antagonist affective to inhibitthe activity of the subject's Y5 receptor, wherein the compound'sbinding to the human Y5 receptor is characterized by a K_(i) less than10 nanomolar when measured in the presence of ¹²⁵I-PYY. In oneembodiment, the compound's binding is characterized by a K_(i) less than1 nanomolar. In another embodiment, the compound's binding to any otherhuman Y-type receptor is characterized by a K_(i) greater than 10nanomolar when measured in the presence of ¹²⁵I-PYY. In anotherembodiment the compound's binding to each of the human Y1, human Y2, andhuman Y4 receptors is characterized by a K_(i) greater than 10 nanomolarwhen measured in the presence of ¹²⁵I-PYY. In a further embodiment, thecompound's binding to any other human Y-type receptor is characterizedby a K_(i) greater than 50 nanomolar. In another embodiment thecompound's binding to any other human Y-type receptor is characterizedby a K_(i) greater than 100 nanomolar. In one embodiment, the compoundbinds to the human Y5 receptor with an affinity greater than ten-foldhigher than the affinity with which the compound binds to any otherhuman Y-type receptor. In another embodiment, the compound binds to thehuman Y5 receptor with an affinity greater than ten-fold higher than theaffinity with which the compound binds to each of the human Y1, humanY2, and human Y4 receptors. The feeding disorder may be obesity orbulimia. The subject may be a vertebrate, a mammal, a human, or a caninesubject.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of anon-peptidyl compound which is a Y5 receptor agonist affective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 100 nanomolar when measured in the presence of ¹²⁵I-PYY;and (b) the binding of the compound to any other human Y-type receptoris characterized by a K_(i) greater than 1000 nanomolar when measured inthe presence of 125I-PYY. In one embodiment, the binding of the compoundto the human Y5 receptor is characterized by a K_(i) less than 10nanomolar.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of anon-peptidyl compound which is a Y5 receptor agonist effective toincrease the activity of the subject's Y5 receptor, wherein (a) thebinding of the compound to the human Y5 receptor is characterized by aK_(i) less than 1 nanomolar when measured in the presence in 125I-PYY;and (b) the compound's binding to any other human Y-type receptor ischaracterized by a K_(i) greater than 100 nanomolar when measured in thepresence of ¹²⁵I-PYY. In one embodiment, the compound binds to the humanY5 receptor with an affinity greater than ten-fold higher than theaffinity with which the compound binds to any other human Y-typereceptor. In another embodiment, the compound binds to the human Y5receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to each of the human Y1, human Y2, andhuman Y4 receptors. The feeding disorder may be anorexia. The subjectmay be a vertebrate, a mammal, a human, or a canine subject.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor agonist effective to increasethe activity of the subject's Y5 receptor, wherein (a) the binding ofthe compound to the human Y5 receptor is characterized by a K_(i) lessthan 1 nanomolar when measured in the presence of ¹²⁵I-PYY; and (b) thebinding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 25 nanomolar when measured in thepresence of 125I-PYY.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor agonist effective to increasethe activity of the subject's Y5 receptor, wherein (a) the binding ofthe compound to the human Y5 receptor is characterized by a K_(i) lessthan 0.1 nanomolar when measured in the presence of ¹²⁵I-PYY; and (b)the binding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 1 nanomolar when measured in thepresence of ¹²⁵I-PYY. In one embodiment, the binding of the agonist toany other human Y-type receptor is characterized by a K_(i) greater than10 nanomolar.

This invention provides a method of treating a feeding disorder in asubject which comprises administering to the subject an amount of apeptidyl compound which is a Y5 receptor agonist effective to increasethe activity of the subject's Y5 receptor, wherein (a) the binding ofthe compound to the human Y5 receptor is characterized by a K_(i) lessthan 0.01 nanomolar when measured in the presence of ¹²⁵I-PYY; and (b)the bonding of the compound to any other human Y-type receptor ischaracterized by a K_(i) greater than 1 nanomolar when measured in thepresence of ¹²⁵I-PYY. In one embodiment, the compound binds to the humanY5 receptor with an affinity greater than ten-fold higher than theaffinity with which the compound binds to any other human Y-typereceptor. In another embodiment, the compound binds to the human Y5receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to each of the human Y1, human Y2, andhuman Y4 receptors. In one embodiment, the feeding disorder is anorexia.The subject may be a vertebrate, a mammal, a human, or a canine subject.

This invention provides an isolated nucleic acid encoding a Y5 receptor.In an embodiment, the Y5 receptor is a vertebrate or a mammalian Y5receptor. In one embodiment, the Y5 receptor is a human Y5 receptor. Inan embodiment, the isolated nucleic acid encodes a receptor beingcharacterized by an amino acid sequence in the transmembrane region,which amino acid sequence has 60% homology or higher to the amino acidsequence in the transmembrane region of the human Y5 receptor shown inFIG. 6. In another embodiment, the Y5 receptor has substantially thesame amino acid sequence as described in FIG. 4. In another embodiment,the Y5 receptor has substantially the same amino acid sequence asdescribed in FIG. 6.

This invention provides the above-described isolated nucleic acid,wherein the nucleic acid is a DNA. In an embodiment, the DNA is a cDNA.In another embodiment, the DNA is a genomic DNA. In still anotherembodiment, the nucleic acid is RNA. In a separate embodiment, thenucleic acid encodes a human Y5 receptor. In an embodiment, the human Y5receptor has the amino acid sequence as described in FIG. 6. In anotherembodiment, the nucleic acid encodes a rat Y5 receptor. In anembodiment, the rat Y5 receptor has the amino acid sequence as shown inFIG. 4. In another embodiment, the nucleic acid encodes a canine Y5receptor. In an embodiment, the canine Y5 receptor has the amino acidsequence shown in FIG. 15.

This invention further provides DNA which is degenerate with any of theDNA shown in FIGS. 3, 5 and 14, wherein the DNA encodes Y5 receptorshaving the amino acid sequences shown in FIGS. 4, 6, and 15,respectively. This invention also encompasses DNAs and cDNAs whichencode amino acid sequences which differ from those of Y5 receptor, butwhich should not produce phenotypic changes. Alternatively, thisinvention also encompasses DNAs and cDNAs which hybridize to the DNA andcDNA of the subject invention. Hybridization methods are well known tothose of skill in the art.

The DNA of the subject invention also include DNA coding for polypeptideanalogs, fragments or derivatives of antigenic polypeptides which differfrom naturally-occurring forms in terms of the identity or location ofone or more amino acid residues (deletion analogs containing less thanall of the residues specified for the protein, substitution analogswherein one or more residues specified are replaced by other residuesand addition analogs where in one or more amino acid residues is addedto a terminal or medial portion of the polypeptides) and which sharesome or all properties of naturally-occurring forms. These nucleic acidsinclude: the incorporation of codons “preferred” for expression byselected non-mammalian hosts; the provision of sites for cleavage byrestriction endonuclease enzymes; and the provision of additionalinitial, terminal or intermediate DNA sequences that facilitateconstruction of readily expressed vectors.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The nucleic acid isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

In a separate embodiment, the nucleic acid encodes a rat Y5 receptor. Inanother embodiment, the rat Y5 receptor has the amino acid sequenceshown in FIG. 4.

This invention also provides an isolated Y5 receptor protein. In oneembodiment, the Y5 receptor protein is a human Y5 receptor protein. Inanother embodiment, the human Y5 receptor protein has the amino acidsequence as shown in FIG. 6. In a further embodiment, the Y5 receptorprotein is a rat Y5 receptor protein. In another embodiment, the rat Y5receptor protein has the amino acid sequence as shown in FIG. 4. Inanother embodiment, the Y5 receptor protein is a canine Y5 receptorprotein. In a further embodiment, the canine Y5 receptor protein has theamino acid sequence as shown in FIG. 15.

This invention provides a vector comprising the above-described nucleicacid.

Vectors which comprise the isolated nucleic acid described hereinabovealso are provided. Suitable vectors comprise, but are not limited to, aplasmid or a virus. These vectors may be transformed into a suitablehost cell to form a host cell vector system for the production of apolypeptide having the biological activity of a Y5 receptor.

This invention provides the above-described vector adapted forexpression in a bacterial cell which further comprises the regulatoryelements necessary for expression of the nucleic acid in the bacterialcell operatively linked to the nucleic acid encoding the Y5 receptor asto permit expression thereof.

This invention provides the above-described vector adapted forexpression in a yeast cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the yeast celloperatively linked to the nucleic acid encoding the Y5 receptor as topermit expression thereof.

This invention provides the above-described vector adapted forexpression in an insect cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the insect celloperatively linked to the nucleic acid encoding the Y5 receptor as topermit expression thereof.

In an embodiment, the vector is adapted for expression in a mammaliancell which comprises the regulatory elements necessary for expression ofthe DNA in the mammalian cell operatively linked to the DNA encoding themammalian Y5 receptor as to permit expression thereof.

In an embodiment, the vector is adapted for expression in a mammaliancell which comprises the regulatory elements necessary for expression ofthe DNA in the mammalian cell operatively linked to the DNA encoding thecanine Y5 receptor as to permit expression thereof.

In a further embodiment, the vector is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the DNA in the mammalian cell operatively linked to theDNA encoding the human Y5 receptor as to permit expression thereof.

In a still further embodiment, the plasmid is adapted for expression ina mammalian cell which comprises the regulatory elements necessary forexpression of the DNA in the mammalian cell operatively linked to theDNA encoding the rat Y5 receptor as to permit expression thereof.

In a still further embodiment, the plasmid is adapted for expression ina mammalian cell which comprises the regulatory elements necessary forexpression of the DNA in the mammalian cell operatively linked to theDNA encoding the canine Y5 receptor as to permit expression thereof.

This invention provides the above-described plasmid adapted forexpression in a mammalian cell which comprises the regulatory elementsnecessary for expression of DNA in a mammalian cell operatively linkedto the DNA encoding the mammalian Y5 receptor as to permit expressionthereof.

This invention provides a plasmid which comprises the regulatoryelements necessary for expression of DNA in a mammalian cell operativelylinked to the DNA encoding the human Y5 receptor as to permit expressionthereof designated pcEXV-hY5 (ATCC Accession No. 75943).

This plasmid (pcEXV-hY5) was deposited on Nov. 4, 1994 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. 75943.

This invention provides a plasmid which comprises the regulatoryelements necessary for expression of DNA in a mammalian cell operativelylinked to the DNA encoding the rat Y5 receptor as to permit expressionthereof designated pcEXV-rY5 (ATCC Accession No. 75944).

This plasmid (pcEXV-rY5) was deposited on Nov. 4, 1994 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. CRL75944.

This invention provides a plasmid designated Y5-bd-5 (ATCC Accession No.______). This invention also provides a plasmid designated Y5-bd-8 (ATCCAccession No. ______). Thses plasmids were deposited on Dec. 1, 1995with the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and were accorded ATCC Accession Nos.______ and ______, respectively.

This invention provides a baculovirus designated hY5-BB3 (ATCC AccessionNo. ______). This baculovirus was deposited on Nov. 15, 1995 with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., 20852, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Accession No. ______.

This invention provides a mammalian cell comprising the above-describedplasmid or vector. In an embodiment, the mammalian cell is a COS-7 cell.

In another embodiment, the mammalian cell is a 293 human embryonickidney cell designated 293-rY5-14 (ATCC Accession No. CRL 11757).

This cell (293-rY5-14) was deposited on Nov. 4, 1994 with the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.20852, U.S.A. under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and was accorded ATCC Accession No. CRL11757.

In a further embodiment, the mammalian cell is a mouse fibroblast (tk−)cell, containing the plasmid pcEXV-hY5 and designated L-hY5-7 (ATCCAccession No. CRL-11995). In another embodiment, the mammalian cell is amouse embryonic NIH-3T3 cell containing the plasmid pcEXV-hY5 anddesignated N-hY5-8 (ATCC Accession No. CRL-11994). These cells weredeposited on Nov. 15, 1995 with the American Type Culture. Collection(ATCC) 12301 Parklawn Drive, Rockville, Md., 20852, U.S.A. under theprovisions ot the Budapest Treaty for the International Recgonition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure, andwere accorded ATCC. Accession Nos. CRL-11995 and CRL-11994,respectively.

This invention provides a nucleic acid probe comprising a nucleic acidmolecule of at least 15 nucleotides capable of specifically hybridizingwith a unique sequence included within the sequence of a nucleic acidmolecule encoding a Y5 receptor. In an embodiment, the nucleic acid isDNA.

This nucleic acid produced can either be DNA or RNA. As used herein, thephrase “specifically hybridizing” means the ability of a nucleic acid torecognize a nucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs.

This nucleic acid of at least 15 nucleotides capable of specificallyhybridizing with a sequence of a nucleic acid encoding the human Y5receptors can be used as a probe. Nucleic acid probe technology is wellknown to those skilled in the art who will readily appreciate that suchprobes may vary greatly in length and may be labeled with a detectablelabel, such as a radioisotope or fluorescent dye, to facilitatedetection of the probe. DNA probe molecules may be produced by insertionof a DNA molecule which encodes the Y5 receptor into suitable vectors,such as plasmids or bacteriophages, followed by transforming intosuitable bacterial host cells, replication in the transformed bacterialhost cells and harvesting of the DNA probes, using methods well known inthe art. Alternatively, probes may be generated chemically from DNAsynthesizers.

RNA probes may be generated by inserting the DNA which encodes the Y5receptor downstream of a bacteriophage promoter such as T3, T7 or SP6.Large amounts of RNA probe may be produced by incubating the labelednucleotides with the linearized fragment where it contains an upstreampromoter in the presence of the appropriate RNA polymerase.

This invention also provides a nucleic acid of at least 15 nucleotidescapable of specifically hybridizing with a sequence of a nucleic acidwhich is complementary to the mammalian nucleic acid encoding a Y5receptor. This nucleic acid may either be a DNA or RNA molecule.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to mRNA encoding a Y5 receptor so asto prevent translation of the mRNA.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to the genomic DNA of a Y5 receptor.

This invention provides an antisense oligonucleotide of a Y5 receptorcomprising chemical analogues of nucleotides.

This invention provides an antibody directed to a Y5 receptor. Thisinvention also provides an antibody directed to a human Y5 receptor.

This invention provides a monoclonal antibody directed to an epitope ofa human Y5 receptor present on the surface of a Y5 receptor expressingcell.

This invention provides a pharmaceutical composition comprising anamount of the oligonucleotide effective to reduce activity of a human Y5receptor by passing through a cell membrane and binding specificallywith mRNA encoding a human Y5 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane. In an embodiment, the oligonucleotide iscoupled to a substance which inactivates mRNA. In another embodiment,the substance which inactivates mRNA is a ribozyme.

This invention provides the above-described pharmaceutical composition,wherein the pharmaceutically acceptable carrier capable of passingthrough a cell membrane comprises a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type.

This invention provides a pharmaceutical composition comprising anamount of an antagonist effective to reduce the activity of a human Y5receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising anamount of an agonist effective to increase activity of a Y5 receptor anda pharmaceutically acceptable carrier.

This invention provides the above-described pharmaceutical compositionwhich comprises an amount of the antibody effective to block binding ofa ligand to the Y5 receptor and a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carriers” means any of thestandard pharmaceutically acceptable carriers. Examples include, but arenot limited to, phosphate buffered saline, physiological saline, waterand emulsions, such as oil/water emulsions.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a human Y5 receptor.

This invention provides a transgenic nonhuman mammal comprising ahomologous recombination knockout of the native Y5 receptor.

This invention provides a transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a human Y5receptor so placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding a Y5 receptor and which hybridizes tomRNA encoding a Y5 receptor thereby reducing its translation.

This invention provides the above-described transgenic nonhuman mammal,wherein the DNA encoding a human Y5 receptor additionally comprises aninducible promoter.

This invention provides the transgenic nonhuman mammal, wherein the DNAencoding a human Y5 receptor additionally comprises tissue specificregulatory elements.

In an embodiment, the transgenic nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of Y5 receptor are produced by creating transgenic animals inwhich the activity of the Y5 receptor is either increased or decreased,or the amino acid sequence of the expressed Y5 receptor is altered, by avariety of techniques. Examples of these techniques include, but are notlimited to: 1) Insertion of normal or mutant versions of DNA encoding aY5 receptor, by microinjection, electroporation, retroviral transfectionor other means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal or 2)Homologous recombination of mutant or normal, human or animal versionsof these genes with the native gene locus in transgenic animals to alterthe regulation of expression or the structure of these Y5 receptorsequences. The technique of homologous recombination is well known inthe art. It replaces the native gene with the inserted gene and so isuseful for producing an animal that cannot express native Y5 receptorsbut does express, for example, an inserted mutant Y5 receptor, which hasreplaced the native Y5 receptor in the animal's genome by recombination,resulting in underexpression of the transporter. Microinjection addsgenes to the genome, but does not remove them, and so is useful forproducing an animal which expresses its own and added Y5 receptors,resulting in overexpression of the Y5 receptors.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding a Y5receptor is purified from a vector by methods well known in the art.Inducible promoters may be fused with the coding region of the DNA toprovide an experimental means to regulate expression of the transgene.Alternatively or in addition, tissue specific regulatory elements may befused with the coding region to permit tissue-specific expression of thetrans-gene. The DNA, in an appropriately buffered solution, is put intoa microinjection needle (which may be made from capillary tubing using apipet puller) and the egg to be injected is put in a depression slide.The needle is inserted into the pronucleus of the egg, and the DNAsolution is injected. The injected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

This invention also provides a method for determining whether a ligandcan specifically bind to a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding the Y5 receptor with theligand under conditions permitting binding of ligands to such receptor,detecting the presence of any such ligand specifically bound to the Y5receptor, and thereby determining whether the ligand specifically bindsto the Y5 receptor.

This invention provides a method for determining whether a ligand canspecifically bind to a human Y5 receptor which comprises contacting acell transfected with and expressing DNA encoding the human Y5 receptorwith the ligand under conditions permitting binding of ligands to suchreceptor, detecting the presence of any such ligand specifically boundto the human Y5 receptor, and thereby determining whether the ligandspecifically binds to the human Y5 receptor.

This invention provides a method for determining whether a ligand canspecifically bind to a human Y5 receptor which comprises contacting acell transfected with and expressing DNA encoding the human Y5 receptorwith the ligand under conditions permitting binding of ligands to suchreceptor, detecting the presence of any such ligand specifically boundto the human Y5 receptor, and thereby determining whether the ligandspecifically binds to the human Y5 receptor, such human Y5 receptorhaving substantially the same amino acid sequence shown in FIG. 6.

This invention provides a method for determining whether a ligand canspecifically bind to a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding the Y5 receptor with theligand under conditions permitting binding of ligands to such receptor,detecting the presence of any such ligand specifically bound to the Y5receptor, and thereby determining whether the ligand specifically bindsto the Y5 receptor, such Y5 receptor being characterized by an aminoacid sequence in the transmembrane region having 60% homology or higherto the amino acid sequence in the transmembrane region of the Y5receptor shown in FIG. 6.

This invention provides a method for determining whether a ligand canspecifically bind to a Y5 receptor which comprises preparing a cellextract from cells transfected with and expressing DNA encoding the Y5receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand under conditionspermitting binding of ligands to such receptor, detecting the presenceof the ligand specifically bound to the Y5 receptor, and therebydetermining whether the ligand specifically binds to the Y5 receptor.

This invention provides a method for determining whether a ligand canspecifically bind to a human Y5 receptor which comprises preparing acell extract from cells transfected with and expressing DNA encoding thehuman Y5 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand under conditionspermitting binding of ligands to the human Y5 receptor, detecting thepresence of the ligand specifically bound to the human Y5 receptor, andthereby determining whether the ligand can specifically bind to thehuman Y5 receptor.

This invention provides a method for determining whether a ligand canspecifically bind to a human Y5 receptor which comprises preparing acell extract from cells transfected with and expressing DNA encoding thehuman Y5 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand under conditionspermitting binding of ligands to the human Y5 receptor, detecting thepresence of the ligand specifically bound to the human Y5 receptor, andthereby determining whether the ligand can specifically bind to thehuman Y5 receptor, such human Y5 receptor having substantially the sameamino acid sequence shown in FIG. 6.

This invention provides a method for determining whether a ligand canspecifically bind to a Y5 receptor which comprises preparing a cellextract from cells transfected with and expressing DNA encoding the Y5receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand under conditionspermitting binding of ligands to the Y5 receptor, detecting the presenceof the ligand specifically bound to the Y5 receptor, and therebydetermining whether the ligand can specifically bind to the Y5 receptor,such Y5 receptor being characterized by an amino acid sequence in thetransmembrane region having 60% homology or higher to the amino acidsequence in the transmembrane region of the Y5 receptor shown in FIG. 6.

In one embodiment, the Y5 receptor is a human Y5 receptor. In anotherembodiment, the Y5 receptor is a rat Y5 receptor. In another embodiment,the Y5 receptor is a canine Y5 receptor.

This invention provides a method for determining whether a ligand is aY5 receptor agonist which comprises contacting a cell transfected withand expressing a Y5 receptor with the ligand under conditions permittingactivation of a functional Y5 receptor response, detecting a functionalincrease in Y5 receptor activity, and thereby determining whether theligand is a Y5 receptor agonist.

This invention provides a method for determining whether a ligand is aY5 receptor agonist which comprises contacting a cell transfected withand expressing a Y5 receptor with the ligand under conditions permittingactivation of the Y5 receptor, detecting an increase in Y5 receptoractivity, and thereby determining whether the ligand is a Y5 receptoragonist.

This invention provides a method-for determining whether a ligand is aY5 receptor agonist which comprises preparing a cell extract from cellstransfected with and expresssing DNA encoding the Y5 receptor, isolatinga membrane fraction from the cell extract, contacting the membranefraction with the ligand under conditions permitting activation of a Y5receptor, and detecting an increase in Y5 receptor activity, so as tothereby determine whether the ligand is a Y5 receptor agonist.

In one embodiment of the above-described methods, the Y5 receptor is ahuman Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5receptor. In a further embodiment, the Y5 receptor is a canine Y5receptor.

This invention provides a method for determining whether a ligand is aY5 receptor antagonist which comprises contacting a cell transfectedwith and expressing DNA encoding a Y5 receptor with the ligand in thepresence of a known Y5 receptor agonist, such as PYY or NPY, underconditions permitting the activation of a functional Y5 receptorresponse, detecting a decrease in Y5 receptor activity, and therebydetermining whether the ligand is a Y5 receptor antagonist.

This invention provides a method for determining whether a ligand is aY5 receptor antagonist which comprises contacting a cell transfectedwith and expressing DNA encoding a Y5 receptor with the ligand in thepresence of a known Y5 receptor agonist, such as PYY or NPY, underconditions permitting the activation of the Y5 receptor, detecting adecrease in Y5 receptor activity, and thereby determining whether theligand is a Y5 receptor antagonist.

This invention provides a method for determining whether a ligand is aY5 receptor antagonist which comprises preparing a cell extract fromcells transfected with and expressing DNA ecoding a Y5 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with the ligand in the presence of a known Y5 receptoragonist, such as PYY or NPY, under conditions permitting the activationof the Y5 receptor, detecting a decrease in Y5 receptor activity, andthereby determining whether the ligand is a Y5 receptor antagonist.

In one embodiment of the above-described methods, the Y5 receptor is ahuman Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5receptor. In a further embodiment, the Y5 receptor is a canine Y5receptor.

In an embodiment of the above-described methods, the cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, 293 human embryonic kidney cell, NIH-3T3 cell orLM(tk-) cell.

In one embodiment of the above-described methods, the ligand is notpreviously known.

This invention provides a Y5 receptor agonist detected by theabove-described method. This invention provides a Y5 receptor antagonistdetected by the above-described method.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a Y5 receptor to identify a compoundwhich specifically binds to the Y5 receptor which comprises (a)contacting a cell transfected with and expressing DNA encoding the Y5receptor with a compound known to bind specifically to the Y5 receptor;(b) contacting the preparation of step (a) with the plurality ofcompounds not known to bind specifically to the Y5 receptor, underconditions permitting binding of compounds known to bind to the Y5receptor; (c) determining whether the binding of the comppound known tobind to the Y5 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to the Y5receptor of each compound included in the plurality of compounds, so asto thereby identify the compound which specifically binds to the Y5receptor.

This invention provides a method of screening a plurality of compoundsnot known to bind to a Y5 receptor to identify a compound whichspecifically binds to the Y5 receptor, which comprises (a) preparing acell extract from cells transfected with and expressing DNA ecoding theY5 recpetor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with a compound known to bindspecifically to the Y5 receptor; (b) contacting the preparation of step(a) with the plurality of compounds not known to bind specifically tothe Y5 receptor, under conditions permitting binding of compounds knownto bind the Y5 receptor; (c) determining whether the binding of thecompound knonw to bind to the Y5 receptor is reduced in the presence ofthe compounds, relative to the binding of the compound in the absnece ofthe plurality of compounds; and if so (d) separately determining thebinding to the Y5 receptor of each compound included in the plurality ofcompounds, so as to thereby-identify the compound which specificallybinds to the Y5 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a Y5 receptor to identify a compoundwhich activates the Y5 receptor which comprises (a) contacting a celltransfected with and expressing the Y5 receptor with the plurality ofcompounds not known to bind specifically to the Y5 receptor, underconditions permitting activation of the Y5 receptor; (b) determiningwhether the activity of the Y5 receptor is increased in the presence ofthe compounds; and if so (c) separately determining whether theactivation of the Y5 receptor is increased by each compound included inthe plurality of compounds, so as to thereby identify the compound whichactivates the Y5 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a Y5 receptor to identify a compoundwhich activates the Y5 receptor which comprises (a) preparing a cellextract from cells transfected with and expressing DNA encoding the Y5receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the plurality of compounds notknown to bind specifically to the Y5 receptor, under conditionspermitting activation of the Y5 receptor; (b) determining whether theactivity of the Y5 receptor is increased in the presence of thecompounds; and if so (c) separately determining whether the activationof the Y5 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the Y5 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a Y5 receptor toidentify a compound which inhibits the activation of the Y5 receptor,which comprises (a) contacting a cell transfected with and expressingthe Y5 receptor with the plurality of compounds in the presence of aknown Y5 receptor agonist, under conditions permitting activation of theY5 receptor; (b) determining whether the activation of the Y5 receptoris reduced in the presence of the plurality of compounds, relative tothe activation of the Y5 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the Y5 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the Y5 receptor.

A method of screening a plurality of chemical compounds not known toinhibit the activation of a Y5 receptor to identify a compound whichinhibits the activation of the Y5 receptor, which comprises (a)preparing a cell extract from cells transfected with and expressing DNAencoding the Y5 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the plurality ofcompounds in the presence of a known Y5 receptor agonist, underconditions permitting activation of the Y5 receptor; (c) separatelydetermining the inhibition of activation of the Y5 receptor for eachcompound included in the plurality of compounds, so as to therebyidentify the compound which inhibits the activation of the Y5 receptor.

In one embodiment of the above-described methods the Y5 receptor is ahuman Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5receptor. In a further embodiment, the Y5 receptor is a canine Y5receptor. In another embodiment, the cell is a mammalian cell. In afurhter embodiment, the mammalian cell is non-neuronal in origin. Thecell may be a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-)cell, or an NIH-3T3 cell.

This invention provides a method of screening drugs to identify drugswhich specifically bind to a Y5 receptor on the surface of a cell whichcomprises contacting a cell transfected with and expressing DNA encodinga Y5 receptor with a plurality of drugs under conditions permittingbinding of drugs to the Y5 receptor, determining those drugs whichspecifically bind to the transfected cell, and thereby identifying drugswhich specifically bind to the Y5 receptor.

This invention provides a method of screening drugs to identify drugswhich act as agonists of a Y5 receptor which comprises contacting a celltransfected with and expressing DNA encoding a Y5 receptor with aplurality of drugs under conditions permitting the activation of afunctional Y5 receptor response, determining those drugs which activatesuch receptor in the cell, and thereby identify drugs which act as Y5receptor agonists. This invention provides a method of screening drugsto identify drugs which act as agonists of a human Y5 receptor whichcomprises contacting a cell transfected with and expressing DNA encodinga human Y5 receptor with a plurality of drugs under conditionspermitting the activation of a functional human Y5 receptor response,determining those drugs which activate such receptor in the cell, andthereby identify drugs which act as human Y5 receptor agonists.

This invention provides a method of screening drugs to identify drugswhich act as Y5 receptor antagonists which comprises contacting cellstransfected with and expressing DNA encoding a Y5 receptor with aplurality of drugs in the presence of a known Y5 receptor agonist, suchas PYY or NPY, under conditions permitting the activation of afunctional Y5 receptor response, determining those drugs which inhibitthe activation of the receptor in the mammalian cell, and therebyidentifying drugs which act as Y5 receptor antagonists.

This invention provides a method of screening drugs to identify drugswhich act as human Y5 receptor antagonists which comprises contactingcells transfected with and expressing DNA encoding a human Y5 receptorwith a plurality of drugs in the presence of a known human Y5 receptoragonist, such as PYY or NPY, under conditions permitting the activationof a functional human Y5 receptor response, determining those drugswhich inhibit the activation of the receptor in the mammalian cell, andthereby identifying drugs which act as human Y5 receptor antagonists. Inan embodiment, the cell is non-neuronal in origin. In a furtherembodiment, the cell is a Cos-7 cell, a 293 human embryonic kidney cell,an LM(tk−) cell or an NIH-3T3 cell.

This invention provides a pharmaceutical composition comprising a drugidentified by the above-described methods and a pharmaceuticallyacceptable carrier. This invention provides a method of detectingexpression of Y5 receptor by detecting the presence of mRNA coding forthe Y5 receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with the above-described nucleic acidprobe under hybridizing conditions, detecting the presence of mRNAhybridized to the probe, and thereby detecting the expression of the Y5receptor by the cell.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of a Y5receptor which comprises administering to a subject an effective amountof the above-described pharmaceutical composition effective to inhibitthe Y5 receptor by the subject.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a Y5 receptorwhich comprises administering to a subject an effective amount of theabove-described pharmaceutical composition effective to activate the Y5receptor in the subject.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of a Y5receptor which comprises administering to a subject an effective amountof Y5 receptor antagonist.

In one embodiment of the above-described methods, the abnormality isobesity. In another embodiment, the abnormality is bulimia.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a Y5 receptorwhich comprises administering to a subject an effective amount of a Y5receptor agonist. In a further embodiment, the abnormal condition isanorexia. In a separate embodiment, the abnormal condition is asexual/reproductive disorder. In another embodiment, the abnormalcondition is depression. In another embodiment, the abnormal conditionis anxiety.

In an embodiment, the abnormal condition is gastric ulcer. In a furtherembodiment, the abnormal condition is memory loss. In a furtherembodiment, the abnormal condition is migraine. In a further embodiment,the abnormal condition is pain. In a further embodiment, the abnormalcondition is epileptic seizure. In a further embodiment, the abnormalcondition is hypertension. In a further embodiment, the abnormalcondition is cerebral hemorrhage. In a further embodiment, the abnormalcondition is shock. In a further embodiment, the abnormal condition iscongestive heart failure. In a further embodiment, the abnormalcondition is sleep disturbance. In a further embodiment, the abnormalcondition is nasal congestion. In a further embodiment, the abnormalcondition is diarrhea.

This invention provides a method of treating obesity in a subject whichcomprises administering to the subject an effective amount of a Y5receptor antagonist.

This invention provides a method of treating anorexia in a subject whichcomprises administering to the subject an effective amount of a Y5receptor agonist.

This invention provides a method of treating bulimia nervosa in asubject which comprises administering to the subject an effective amountof a Y5 receptor antagonist. This invention provides a method ofinducing a subject to eat which comprises administering to the subjectan effective amount of a Y5 receptor agonist. In one embodiment, thesubject is a vertebrate. In another embodiment, the subject is a human.In another embodiment, the subject is a rat. In another embodiment, thesubject is a canine subject.

This invention provides a method of increasing the consumption of a foodproduct by a subject which comprises a composition of the food productand an effective amount of a Y5 receptor agonist. In one embodiment, thesubject is a vertebrate. In another embodiment, the subject is a human,a rat or a canine subject.

This invention provides a method of treating abnormalities which arealleviated by reduction of activity of a human Y5 receptor whichcomprises administering to a subject an amount of the above-describedpharmaceutical composition effective to reduce the activity of human Y5receptor and thereby alleviate abnormalities resulting from overactivityof a human Y5 receptor.

This invention provides a method of treating an abnormal conditionrelated to an excess of Y5 receptor activity which comprisesadministering to a subject an amount of the pharmaceutical compositioneffective to block binding of a ligand to the Y5 receptor and therebyalleviate the abnormal condition.

This invention provides a method of detecting the presence of a human Y5receptor on the surface of a cell which comprises contacting the cellwith the antibody capable of binding to the human Y5 receptor underconditions permitting binding of the antibody to the receptor, detectingthe presence of the antibody bound to the cell, and thereby detectingthe presence of a human Y5 receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of a human Y5 receptors whichcomprises producing a transgenic nonhuman mammal whose levels of humanY5 receptor activity are varied by use of an inducible promoter whichregulates human Y5 receptor expression.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of a human Y5 receptors whichcomprises producing a panel of transgenic nonhuman mammals eachexpressing a different amount of human Y5 receptor.

This invention provides a method for identifying a substance capable ofalleviating the abnormalities resulting from overactivity of a human Y5receptor comprising administering a substance to the above-describedtransgenic nonhuman mammals, and determining whether the substancealleviates the physical and behavioral abnormalities displayed by thetransgenic nonhuman mammal as a result of overactivity of a human Y5receptor.

This invention provides a method for treating the abnormalitiesresulting from overactivity of a human Y5 receptor which comprisesadministering to a subject an amount of the above-describedpharmaceutical composition effective to alleviate the abnormalitiesresulting from overactivity of a human Y5 receptor.

This invention provides a method for identifying a substance capable ofalleviating the abnormalities resulting from underactivity of a human Y5receptor comprising administering the substance to the above-describedtransgenic nonhuman mammals and determining whether the substancealleviates the physical and behavioral abnormalities displayed by thetransgenic nonhuman mammal as a result of underactivity of a human Y5receptor.

This invention provides a method for treating the abnormalitiesresulting from underactivity of a human Y5 receptor which comprisesadministering to a subject an amount of the above-describedpharmaceutical composition effective to alleviate the abnormalitiesresulting from underactivity of a human Y5 receptor.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific human Y5 receptorallele which comprises: a obtaining DNA of subjects suffering from thedisorder; performing a restriction digest of the DNA with a panel ofrestriction enzymes; c. electrophoretic-ally separating the resultingDNA fragments on a sizing gel; d. contacting the resulting gel with anucleic acid probe capable of specifically hybridizing to DNA encoding ahuman Y5 receptor and labelled with a detectable marker; e. detectinglabelled bands which have hybridized to the DNA encoding a human Y5receptor labelled with a detectable marker to create a unique bandpattern specific to the DNA of subjects suffering from the disorder; f.preparing DNA obtained for diagnosis by steps a-e; and g. comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step e and the DNA obtained for diagnosis from step f todetermine whether the patterns are the same or different and to diagnosethereby predisposition to the disorder if the patterns are the same. Inan embodiment, a disorder associated with the activity of a specifichuman Y5 receptor allele is diagnosed.

This invention provides a method of preparing an isolated Y5 receptorwhich comprises: a. inducing cells to express the Y5 receptor; b.recovering the receptor from the resulting cells; and c. purifying thereceptor so recovered.

This invention provides a method of preparing the isolated Y5 receptorwhich comprises: a. inserting nucleic acid encoding Y5 receptor in asuitable vector adapted for expression in a bacterial, yeast insect ormammalian cell operatively linked to the nucleic acid encoding the Y5receptor as to permit expression thereof; b. inserting the resultingvector in a suitable host cell so as to obtain a cell which produces theY5 receptor; c. recovering the receptor produced by the resulting cell;and d. purifying the receptor so recovered.

This invention well be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

Materials and Methods

cDNA Cloning

Total RNA was prepared by a modification of the guanidine thiocyanatemethod (Kingston, 1987), from 5 grams of rat hypothalamus (Rockland,Gilbertsville, Pa.). Poly A⁺RNA was purified with a FastTrack kit(Invitrogen Corp., San Diego, Calif.). Double stranded (ds) cDNA wassynthesized from 7 μg of poly A⁺ RNA according to Gubler and Hoffman(Gubler and Hoffman, 1983), except that ligase was omitted in the secondstrand cDNA synthesis. The resulting DS cDNA was ligated to BstxI/EcoRIadaptors (Invitrogen Corp.), the excess of adaptors was removed bychromatography on Sephacryl 500 HR (Pharmacia®-LKB) and the ds-cDNA sizeselected on a Gen-Pak Fax HPLC column (Millipore Corp., Milford, Mass.).High molecular weight fractions were ligated in pEXJ.BS (A cDNA cloningexpression vector derived from pcEXV-3; Okayama and Berg, 1983; Millerand Germain, 1986) cut by BstxI as described by Aruffo and Seed (Aruffoand Seed, 1987). The ligated DNA was electroporated in E. Coli MC 1061F⁺ (Gene Pulser, Biorad). A total of 3.4×10⁶ independent clones with aninsert mean size of 2.7 kb could be generated. The library was plated onPetri dishes (Ampicillin selection) in pools of 6.9 to 8.2×10³independent clones. After 18 hours amplification, the bacteria from eachpool were scraped, resuspended in 4 mL of LB media and 1.5 mL processedfor plasmid purification with a QIAprep-8 plasmid kit (Qiagen Inc,Chatsworth, Calif.). 1 ml aliquots of each bacterial pool were stored at−85° C. in 20% glycerol.

Isolation of a cDNA Clone Encoding an Atypical Rat Hypothalamic NPY5Receptor

DNA from pools of 7500 independent clones was transfected into COS-7cells by a modification of the DEAE-dextran procedure (Warden andThorne, 1968). COS-7 cells were grown in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, 2 mM L-glutamine (DMEM-C) at 37°C. in 5% CO₂. The cells were seeded one day before transfection at adensity of 30,000 cells/cm² on Lab-Tek chamber slides (1 chamber,Permanox slide from Nunc Inc., Naperville, Ill.). On the next day, cellswere washed twice with PBS, 735 μl of transfection cocktail was addedcontaining 1/10 of the DNA from each pool and DEAE-dextran (500 μg/ml)in Opti-MEM I serum free media (Gibco BRL LifeTechnologies Inc. GrandIsland, N.Y.). After a 30 min. incubation at 37° C., 3 ml of chloroquine(80 μM in DMEM-C) was added and the cells incubated a further 2.5 hoursat 37° C. The media was aspirated from each chamber and 2 ml of 10% DMSOin DMEM-C added. After 2.5 minutes incubation at room temperature, themedia was aspirated, each chamber washed once with 2 ml PBS, the cellsincubated 48 hours in DMEM-C and the binding assay was performed on theslides. After one wash with PBS, positive pools were identified byincubating the cells with 1 nM (3×10⁶ cpm per slide) of porcine[¹²⁵I]-PYY (NEN; SA=2200 Ci/mmole) in 20 mM Hepes-NaOH pH 7.4, CaCl21.26 mM, MgSO4 0.81 mM, KH₂PO₄ 0.44 mM, KCL 5.4, NaCl 10 mM, 0.1% BSA,0.1% bacitracin for 1 hour at room temperature. After six washes (threeseconds each) in binding buffer without ligand, the monolayers werefixed in 2.5% glutaraldehyde in PBS for five minutes, washed twice fortwo minutes in PBS, dehydrated in ethanol baths for two minutes each(70, 80, 95, 100%) and air dried. The slides were then dipped in 100%photoemulsion (Kodak type NTB2) at 42° C. and exposed in the dark for 48hours at 4° C. in light proof boxes containing drierite. Slides weredeveloped for three minutes in Kodak D19 developer (32 g/l of water),rinsed in water, fixed in Kodak fixer for 5 minutes, rinsed in water,air dried and mounted with 5 Aqua-Mount (Lerner Laboratories,Pittsburgh, Pa.). Slides were screened at 25× total magnification. Asingle clone, CG-18, was isolated by SIB selection as described (McCormick, 1987). DS-DNA was sequenced with a Sequenase kit (USBiochemical, Cleveland, Ohio) according to the manufacturer. Nucleotideand peptide sequence analysis were performed with GCG programs (GeneticsComputer group, Madison, Wis.).

Isolation of the Human Y5 Homolog

Using rat oligonucleotide primers in TM 3 (sense primer; position484-509 in FIG. 1A) and in TM 6 (antisense primer; position 1219-1243 inFIG. 3A), applicants screened a human hippocampal cDNA library using thepolymerase chain reaction. 1 μl (4×10⁶ bacteria) of each of 450amplified pools containing each ≈5000 independent clones andrepresenting a total of 2.2×10⁶ was subjected directly to 40 cycles ofPCR and the resulting products analyzed by agarose gel electrophoresis.One of three positive pools was analyzed further and by sib selection asingle cDNA clone was isolated and characterized. This cDNA turned outto be full length and in the correct orientation for expression. DS-DNAwas sequenced with a sequenase kit (US Biochemical, Cleveland, Ohio)according to the manufacturer.

Isolation of the Canine Y5 Homolog

An alignment of the coding nucleotide sequences of the rat and human Y5receptors was used to synthesize a pair of PCR primers. A regionupstream of TM III which is 100% conserved between rat and human waschosen to synthesize the forward primer CH 156:5′-TGGATCAGTGGATGTTTGGCAAAG-3′ (Seq. I.D. No. 7).

A region at the carboxy end of the 5-6 loop, immediately. upstream ofTM6, which is also 100% conserved between rat and human sequences waschosen to synthesize the reverse primer CH153: (Seq. I.D. No. 8)5′-GTCTGTAGAAAACACTTCGAGATCTCTT-3′.

The primers CH156-CH153 were used to amplify 10 ng of poly (A+) RNA fromrat brain that was reverse transcribed using the SSII reversetranscriptase (GibcoBRL, Gaithersburg, Md.). PCR was performed onsingle-stranded cDNA with Taq Polymerase (Perkin Elmer-Roche MolecularSystems, Branchburg, N.J.)under the following conditions: 94° C. for 1min, 60° C. for 1 min and 72° C. for 1 min for 40 cycles The resulting798 bp PCR DNA fragment was subcloned in pCR Script (Stratagene, LaJolla, Calif.) and sequenced using a sequenase kit (USB, Cleveland,Ohio) and is designated Y5-bd-5.

3′ and 5′ RACE

The missing 3′ and 5′ ends of the beagle dog Y5 receptor sequences wereisolated by 3′ and 5′ RACE using a Marathon cDNA amplification kit(Clontech, Palo Alto, Calif.). From the sequence of the beagle dog PCRDNA fragment described above, the following PCR primers weresynthesized: (3′ RACE) CH 204: (Seq. I.D. No. 9)5′-CTTCCAGTGTTTCACAGTCTGGTGG-3′; CH 218 (nested primer): (Seq. I.D. No.10) 5′-CTGAGCAGCAGGTATTTATGTGTTG-3′; (5′ RACE) CH 219: (Seq. I.D. No.11) 5′-CTGGATGAAGAATGCTGACTTCTTACAG-3′; CH 245 (nested primer): (Seq.I.D. No. 12) 5′-TTCTTGAGTGGTTCTCTTGAGGAGG-3′.

The 3′ and 5′ RACE reactions were carried out on beagle dog thalamiccDNA according to the kit specifications, with the primers describedabove. The resulting PCR DNA products (smear of 0.7 to 10 kb) werepurified from an agarose gel and reamplified using the nested primersdescribed above. The resulting discrete DNA bands were again purifiedfrom an agarose gel and subcloned in pCR Script (Stratagene, La Jolla,Calif.).

The nucleotide sequence corresponding to the 3′ end of the cDNA wasdetermined and the plasmid designated Y5-bd-8. The nucleotide sequencecorresponding to the 5′ end will be determined in the near future. Thosenucleotide sequences will then be used to synthesize exact primersagainst the initiation and stop codon regions and those exact primerswill then be used to amplify canine thalamic cDNA to generate a PCRproduct corresponding to the full length coding region of the canine Y5receptor, using the Expand High Fidelity polymerase (Boehringer MannheimCorporation, Indianapolis, Ind.). The resulting PCR DNA product will besubcloned in the expression vector pEXJ and the entire coding region ofthe canine Y5 nucleotide sequence will be determined using a SequenaseKit (USB, Cleveland, Ohio).

Northern Blots

Human brain multiple tissue northern blots (MTN blots II and III,Clontech, Palo Alto, Calif.) carrying mRNA purified from various humanbrain areas was hybridized at high stringency according to themanufacturer specifications. The probe was a 0.8 kb DNA PCR fragmentcorresponding to the TM III-carboxy end of the 5-6 loop in the codingregion of the human Y5 receptor subtype.

A rat multiple tissue northern blot (rat MTN blot, Clontech, Palo Alto,Calif.) carrying mRNA purified from various rat tissues was hybridizedat high stringency according to the manufacturer specifications. Theprobe was a 0.8 kb DNA PCR fragment corresponding to the TM III-carboxyend of the 5-6 loop in the coding region of the rat Y5 receptor subtype.

Southern Blot

Southern blots (Geno-Blot, clontech, Palo Alto, Calif.) containing humanor rat genomic DNA cut with five different enzymes (8 μg DNA per lane)was hybridized at high stringency according to the manufacturerspecifications. The probe was a 0.8 kb DNA PCR fragment corresponding tothe TM III-carboxy end of the 5-6 loop in the coding region of the humanand rat Y5 receptor subtypes.

Production of Recombinant Baculovirus

A BamHI site directly 5′ to the starting methionine of human Y5 wasgenetically engineered by replacing the beginning ≈100 base pairs of hY5(i.e. from the starting methionine to an internal EcoRI site) with twooverlapping synthetically-derived oligonucleotides (≈100 bases each),containing a 5′ BamHI site and a 3′ EcoRI site. This permitted theisolation of an ≈1.5 kb Bam HI/Hind III fragment containing the codingregion of hY5. This fragment was subcloned into pBlueBacIII™ into theBam HI/Hind III sites found in the polylinker (construct calledpBB/hY5). To generate baculovirus, 0.5 μg of viral DNA (BaculoGold™) and3 μg of pBB/hY5 were co-transfected into 2×10⁶ Spodoptera frugiperdainsect Sf9 cells by calcium phosphate co-precipitation method, asoutlined in by Pharmingen (in “Baculovirus Expression Vector System:Procedures and Methods Manual”). The cells were incubated for 5 days at27° C. The supernatant of the co-transfection plate was collected bycentrifugation and the recombinant virus (hY5BB3) was plaque purified.The procedure to infect cells with virus, to prepare stocks of virus andto titer the virus stocks were as described in Pharmingen's manual.

Cell Culture

COS-7 cells were grown on 150 mm plates in D-MEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells were trypsinized and split 1:6 every3-4 days. Human embryonic kidney 293 cells were grown on 150 mm platesin D-MEM with supplements (minimal essential medium) with Hanks' saltsand supplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/ml penicillin/100 μg/ml streptomycin)at 37° C., 5% CO₂. Stock plates of 293 cells were trypsinized and split1:6 every 3-4 days. Mouse fibroblast LM(tk−) cells were grown on 150 mmplates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO₂. Stock plates of LM(tk−) cells weretrypsinized and split 1:10 every 3-4 days.

LM(tk−) cells stably transfected with the human Y5 receptor wereroutinely converted from an adherent monolayer to a viable suspension.Adherent cells were harvested with trypsin at the point of confluence,resuspended in a minimal volume of complete DMEM for a cell count, andfurther diluted to a concentration of 10⁶ cells/ml in suspension media(10% bovine calf serum, 10% 10× Medium 199 (Gibco), 9 mM NaHCO₃, 25 mMglucose, 2 mM L-glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin, and 0.05% methyl cellulose). The cell suspension wasmaintained in a shaking incubator at 37° C., 5% CO₂ for 24 hours.Membranes harvested from cells grown in this manner may be stored aslarge, uniform batches in liquid nitrogen. Alternatively, cells may bereturned to adherent cell culture in complete DMEM by distribution into96-well microtiter plates coated with poly-D-lysine (0.01 mg/ml)followed by incubation at 37° C., 5% CO₂ for 24 hours. Cells prepared inthis manner yielded a robust and reliable NPY-dependent response in cAMPradio-immunoassays as further described hereinbelow.

Mouse embryonic fibroblast NIH-3T3 cells were grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells weretrypsinized and split 1:15 every 3-4 days.

Sf9 and Sf21 cells were grown in monolayers on 150 mm tissue culturedishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C., no CO₂. High Five insect cells were grown on 150 mm tissue culturedishes in Ex-Cell 400™ medium supplemented with L-Glutamine, also at 27°C., no CO₂.

Transient Transfection

All receptor subtypes studied (human and rat Y1, human and rat Y2, humanand rat Y4, human and rat Y5) were transiently transfected into COS-7cells by the DEAE-dextran method, using 1 μg of DNA/10⁶ cells (Cullen,1987). The Y1 recepotr was prepared using to known methods (Larhammar,et al., 1992).

Stable Transfection

Human Y1, human Y2, and rat Y5 receptors were co-transfected with aG-418 resistant gene into the human embryonic kidney 293 cell line by acalcium phosphate transfection method (Cullen, 1987). Stably transfectedcells were selected with G-418. Human Y4 and human Y5 receptors weresimilarly transfected into mouse fibroblast LM(tk−) cells and NIH-3T3cells.

Expression of other G-protein coupled receptors

α₁ Human Adrenergic Receptors: To determine the binding of compounds tohuman α₁ receptors, LM(tk−) cell lines stably transfected with the genesencoding the α_(1a), α_(1b), and α_(1d) receptors were used. Thenomenclature describing the α₁ receptors was changed recently, such thatthe receptor formerly designated α_(1a) is now designated α_(1d), andthe receptor formerly designated α_(1c), is now designated α_(1a) (ref).The cell lines expressing these receptors were deposited with the ATCCbefore the nomenclature change and reflect the subtype desgnationsformerly assigned to these receptors. Thus, the cell line expressing thereceptor described herein as the α_(1a) receptor was deposited with theATCC on Sep. 25, 1992, under ATCC Accession No. CRL 11140 with thedesignation L-α_(1c). The cell line expressing receptor described hereinas the α_(1d) receptor was deposited with the ATCC on Sep. 25, 1992,under ATCC Accession No. CRL 11138 with the designation L-α_(1A). Thecell line expressing the α_(1b) receptor is designated L-α_(1B), and wasdeposited on Sep. 25, 1992, under ATCC Accession No. CRL 11139.

α₂ Human Adrenergic Receptors: To determine the binding of compounds tohuman α₂ receptors, LM(tk−) cell lines stably transfected with the genesencoding the α_(2A), α_(2B), and α_(2C) receptors were used. The cellline expressing the 2A receptor is designated L-α_(2A) and was depositedon Nov. 6, 1992, under ATCC Accession No. CRL 11180. The cell lineexpressing the α_(2B) receptor is designated L-NGC-α_(2B), and wasdeposited on Oct. 25, 1989, under ATCC Accession No. CRL 10275. The cellline expressing the α_(2C) receptor is designated L-α_(2C), and wasdeposited on Nov. 6, 1992, under ATCC Accession No. CRL-11181. Celllysates were prepared as described below (see Radioligand Binding toMembrane Suspensions), and suspended in 25 mM glycylglycine buffer (pH7.6 at room temperature). Equilibrium competition binding assay wereperformed using [³H]rauwolscine (0.5 nM), and nonspecific binding wasdetermined by incubation with 10 μM phentolamine. The bound radioligandwas separated by filtration through GF/B filters using a cell harvester.

Human Histamine H₁Receptor: The coding sequence of the human histamineH₁ receptor, homologous to the bovine H₁ receptor, was obtained from ahuman hippocampal cDNA library, and was cloned into the eukaryoticexpression vector pcEXV-3. The plasmid DNA for the H₁ receptor isdesignated pcEXV-H1, and was deposited on Nov. 6, 1992, under ATCCAccession No. 75346. This construct was transfected into COS-7 cells bythe DEAE-dextran method. Cells were harvested after 72 hours and lysedby sonication in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. The cell lysates werecentrifuged at 1000 rpm for 5 min at 4° C., and the supernatant wascentrifuged at 30,000×g for 20 min. at 4° C. The pellet was suspended in37.8 mM NaHPO₄, 12.2 mM KH₂PO₄, pH 7.5. The binding of the histamine H₁antagonist [³H]mepyramine (1 nM, specific activity: 24.8 Ci/mM) was donein a final volume of 0.25 mL and incubated at room temperature for 60min. Nonspecific binding was determined in the presence of 10 μMmepyramine. The bound radioligand was separated by filtration throughGF/B filters using a cell harvester.

Human Histamine H₂ Receptor: The coding sequence of the human H₂receptor was obtained from a human placenta genomic library, and clonedinto the cloning site of PCEXV-3 eukaryotic expression vector. Theplasmid DNA for the H₂ receptor is designated pcEXV-H2, and wasdeposited on Nov. 6, 1992 under ATCC Accession No. 75345. This constructwas transfected into COS-7 cells by the DEAE-dextran method. Cells wereharvested after 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mMEDTA, pH 7.5. The cell lysates were centrifuged at 1000 rpm for 5 min at4° C., and the supernatant was centrifuged at 30,000×g for 20 min at 4°C. The pellet was suspended in 37.8 mM NaHPO₄, 12.2 mM K₂PO₄, pH 7.5.The binding of the histamine H₂ antagonist [³H]tiotidine (5 nM, specificactivity: 70 Ci/mM) was done in a final volume of 0.25 ml and incubatedat room temperature for 60 min. Nonspecific binding was determined inthe presence of 10 μM histamine. The bound radioligand was separated byfiltration through GF/B filters using a cell harvester.

Human Serotonin Receptors:

5HT_(1Dα), 5HT_(1Dβ), 5HT_(1E), 5HT_(1F) Receptors: LM(tk−) clonal celllines stably transfected with the genes encoding each of these 5HTreceptor subtypes were prepared as described above. The cell line forthe 5HT_(1Dα) receptor, designated as Ltk-8-30-84, was deposited on Apr.17, 1990, and accorded ATCC Accession No. CRL 10421. The cell for the5HT_(1Dβ)receptor, designated as Ltk-11, was deposited on Apr. 17,1990,—and accorded ATCC Accession No. CRL 10422. The cell line for the5HT_(1E) receptor, designated 5 HT_(1E)-7, was deposited on Nov. 6,1991, and accorded ATCC Accession No. CRL 10913. The cell line for the5HT_(1F) receptor, designated L-5-HT_(1F), was deposited on Dec. 27,1991, and accorded ATCC Accession No. ATCC 10957. Membrane preparationscomprising these receptors were prepared as described below, andsuspended in 50 mM Tris-HCl buffer (pH 7.4 at 37° C.) containing 10 mMMgCl₂, 0.2 mM EDTA, 10 μM pargyline, and 0.1% ascorbate. The binding ofcompounds was determined in competition binding assays by incubation for30 minutes at 37° C. in the presence of 5nM [³H] serotonin. Nonspecificbinding was determined in the presence of 10 μM serotonin. The boundradioligand was separated by filtration through GF/B filters using acell harvester.

Human 5HT₂ Receptor: The coding sequence of the human 5HT₂ receptor wasobtained from a human brain cortex cDNA library, and cloned into thecloning site of pcEXV-3 eukaryotic expression vector. This construct wastransfected into COS-7 cells by the DEAE-dextran method. Cells wereharvested after 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mMEDTA, pH 7.5. This cell line was deposited with the ATCC on Oct. 31,1989, designated as L-NGC-5HT₂, and was accorded ATCC Accession No. CRL10287. The cell lysates were centrifuged at 1000 rpm for 5 minutes at 4°C., and the supernatant was centrifuged at 30,000×g for 20 minutes at 4°C. The pellet was suspended in 50 mM Tris-HCl buffer (pH 7.7 at roomtemperature) containing 10 mM MgSO₄, 0.5mM EDTA, and 0.1% ascorbate. Thepotency of alpha-1 antagonists at 5HT₂ receptors was determined inequilibrium competition binding assays using [3H]ketanserin (1 nM).Nonspecific binding was defined by the addition of 10 μM mianserin. Thebound radioligand was separated by filtration through GF/B filters usinga cell harvester.

Human 5-HT₇ Receptor: A LM(tk−) clonal cell line stably transfected withthe gene encoding the 5HT₇ receptor subtype was prepared as describedabove. The cell line for the 5HT₇ receptor, designated as L-5HT_(4B),was deposited on Oct. 20, 1992, and accorded ATCC Accession No. CRL11166.

Human Dopamine D₃ Receptor: The binding of compounds to the human D3receptor was determined using membrane preparations from COS-7 cellstransfected with the gene encoding the human D₃ receptor. The humandopamine D3 receptor was prepared using known methods. Sokoloff, P. etal., Nature, 347, 146 (1990), and deposited with the European MolecularBiological Labora-tory (EMBL) Genbank as X53944). Cells were harvestedafter 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mM EDTA, pH7.5. The cell lysates were centrifuged at 1000 rpm for 5 minutes at 4°C., and the supernatant was centrifuged at 30,000×g for 20 minutes at 4°C. The pellet was suspended in 50 mM Tris-HCl (pH 7.4) containing 1 mMEDTA, 5 mM KCl, 1.5 mM CaCl₂, 4mM MgCl₂, and 0.1% ascorbic acid. Thecell lysates were incubated with [³H] spiperone (2 nM), using 10 μM(+)Butaclamol to determine nonspecific binding.

Membrane Harvest

Membranes were harvested from COS-7 cells 48 hours after transienttransfection. Adherent cells were washed twice in ice-cold phosphatebuffered saline (138 mM NaCl, 8.1 mM Na₂HPO₄, 2.5 mM KCl, 1.2 mM KH₂PO₄,0.9 mM CaCl₂, 0.5 mM MgCl₂₁ pH 7.4) and lysed by sonication in ice-coldsonication buffer (20 mM Tris-HCl, 5 mM EDTA, pH 7.7). Large particlesand debris were cleared by low speed centrifugation (200×g, 5 min, 4°C.). Membranes were collected from the supernatant fraction bycentrifugation (32,000×g, 18 min, 4° C.), washed with ice-cold hypotonicbuffer, and collected again by centrifugation (32,000×g, 18 min, 4° C.).The final membrane pellet was resuspended by sonication into a smallvolume of ice-cold binding buffer (˜1 ml for every 5 plates: 10 mM NaCl,20 mM HEPES, 0.22 mM KH₂PO₄, 1.26 mM CaCl₂, 0.81 mM MgSO₄, pH 7.4).Protein concentration was measured by the Bradford method (Bradford,1976) using Bio-Rad Reagent, with bovine serum albumin as a standard.Membranes were held on ice for up to one hour and used fresh, orflash-frozen and stored in liquid nitrogen.

Membranes were prepared similarly from 293, LM(tk−), and NIH-3T3 cells.To prepare membranes from baculovirus infected cells, 2×10⁷ Sf21 cellswere grown in 150 mm tissue culture dishes and infected with ahigh-titer stock of hY5BB3. Cells were incubated for 2-4 days at 27° C.,no CO₂ before harvesting and membrane preparation as described above.

Membranes were prepared similarly from dissected rat hypothalamus.Frozen hypothalami were homogenized for 20 seconds in ice-coldsonication buffer with the narrow probe of a Virtishear homogenizer at1000 rpm (Virtis, Gardiner, N.Y.). Large particles and debris werecleared by centrifugation (200×g, 5 min, 420 C.) and the supernatantfraction was reserved on ice. Membranes were further extracted from thepellet by repeating the homogenization and centrifugation procedure twomore times. The supernatant fractions were pooled and subjected to highspeed centrifugation (100,000×g, 20 min. 4° C.). The final membranepellet was resuspended by gentle homogenization into a small volume ofice-cold binding buffer (1 mL/gram wet weight tissue) and held on icefor up to one hour, —or flash-frozen and stored in liquid nitrogen.

Radioligand Binding to Membrane Suspensions

Membrane suspensions were diluted in binding buffer supplemented with0.1% bovine serum albumin to yield an optimal membrane proteinconcentration so that 125I-PYY (or alternative radioligand such as¹²⁵I-NPY, ¹²⁵I-PYY₃₋₃₆, or ¹²⁵I-[Leu³¹Pro³⁴]PYY) bound by membranes inthe assay was less than 10% of ¹²⁵I-PYY (or alternative radioligand)delivered to the sample (100,000 dpm/sample=0.08 nM for competitionbinding assays). ¹²⁵I-PYY (or alternative radioligand) and peptidecompetitors were also diluted to desired concentrations in supplementedbinding buffer. Individual samples were then prepared in 96-wellpolypropylene microtiter plates by mixing ¹²⁵I-PYY (25 μL) (oralternative radioligand), competing peptides or supplemented bindingbuffer (25 μL), and finally, membrane suspensions (200 μl). Samples wereincubated in a 30° C. water bath with constant shaking for 120 min.

Incubations were terminated by filtration over Whatman GF/C filters(pre-coated with 1% polyethyleneimine and air-dried before use),followed by washing with 5 mL of ice-cold binding buffer. Filter-trappedmembranes were impregnated with MultiLex solid scintillant (Wallac,Turku, Finland) and counted for ¹²⁵I in a Wallac Beta-Plate Reader.Non-specific binding was defined by 300 nM human NPY for all receptorsexcept the Y4 subtypes; 100 nM human PP was used for the human Y4 and100 nM rat PP for the rat Y4. Specific binding in time course andcompetition studies was typically 80%; most non-specific binding wasassociated with the filter. Binding data were analyzed using nonlinearregression and statistical techniques available in the GraphPAD Prismpackage (San Diego, Calif.).

Functional Assay: Radioimmunoassay of cAMP

Stably transfected cells were seeded into 96-well microtiter plates andcultured until confluent. To reduce the potential for receptordesensitization, the serum component of the media was reduced to 1.5%for 4 to 16 hours before the assay. Cells were washed in Hank's bufferedsaline, or HBS (150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 5 mM KCl, 1 mMMgCl₂, and 10 mM glucose) supplemented with 0.1% bovine serum albuminplus 5 mM theophylline and pre-equilibrated in the same solution for 20min at 37° C. in 5% CO₂. Cells were then incubated 5 min with 10 μMforskolin and various concentrations of receptor-selective ligands. Theassay was terminated by the removal of HBS and acidification of thecells with 100 mM HCl. Intracellular cAMP was extracted and quantifiedwith a modified version of a magnetic bead-based radioimmunoassay(Advanced Magnetics, Cambridge, Mass.). The final antigen/antibodycomplex was separated from free ¹²⁵I-cAMP by vacuum filtration through aPVDF filter in a microtiter plate (Millipore, Bedford, Mass.). Filterswere punched and counted for ¹²⁵I in a Packard gamma counter. Bindingdata were analyzed using nonlinear regression and statistical techniquesavailable in the GraphPAD Prism package. (San Diego, Calif.).

Functional Assay: Intracellular Calcium Mobilization

The intracellular free calcium concentration was measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(ref). Stably transfected cells were seeded onto a 35 mm culture dishcontaining a glass coverslip insert. Cells were washed with and loadedwith 100 μl of Fura-2/AM (10 μM) for 20 to 40 min. After washing withHBS to remove the Fura-2/AM solution, cells were equilibrated in HBS for10 to 20 min. Cells were then visualized under the 40× objective of aLeitz Fluovert-FS microscope and fluorescence emission was determined at510 nM with excitation wave lengths alternating between 340 nM and 380nM. Raw fluorescence data were converted to calcium concentrations usingstandard calcium concentration curves and software analysis techniques.

Tissue Preparation for Neuroanatomical Studies

Male Sprague-Dawley rats (Charles Rivers) were decapitated and thebrains rapidly removed and frozen in isopentane. Coronal sections werecut at 11 μm on a cryostat and thaw-mounted onto poly-L-lysine coatedslides and stored at −80° C. until use. Prior to hybridization, tissueswere fixed in 4% paraformaldehyde, treated with 5 mM dithiothreitol,acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride,delipidated with chloroform, and dehydrated in graded ethanols.

Probes

The oligonucleotide probes employed to characterize the distribution ofthe rat NPY Y5 mRNA were complementary to nucleotides 1121 to 1165 inthe 5,6-loop of the rat Y5 mRNA (FIG. 3A) 45 mer antisense and senseoligonucleotide probes were synthesized on a Millipore Expedite 8909Nucleic Acid Synthesis System. The probes were then lyophilized,reconstituted in sterile water, and purified on a 12% polyacrylamidedenaturing gel. The purified probes were again reconstituted to aconcentration of 100 ng/μl, and stored at −20° C.

In Situ Hybridization

Probes were 3′-end labeled with ³⁵S-DATP (1200 Ci/mmol, New EnglandNuclear, Boston, Mass.) to a specific activity of 10⁹ dpm/μg usingterminal deoxynucleotidyl transferase (Pharmacia). The radiolabeledprobes were purified on Biospin 6 chromatography columns (Bio-Rad;Richmond, Calif.), and diluted in hybridization buffer to aconcentration of 1.5×10⁴ cpm/μl. The hybridization buffer consisted of50% formamide, 4× sodium citrate buffer (1×SSC=0.15 M NaCl and 0.015 Msodium citrate), 1× Denhardt's solution (0.2% polyvinylpyrrolidine, 0.2%Ficoll, 0.2% bovine serum albumin), 50 mM dithiothreitol, 0.5 mg/mlsalmon sperm DNA, 0.5 mg/ml yeast tRNA, and 10% dextran sulfate. Onehundred μl of the diluted radiolabeled probe was applied to eachsection, which was then covered with a Parafilm coverslip. Hybridizationwas carried out overnight in humid chambers at 40 to 55° C. Thefollowing day the sections were washed in two changes of 2×SSC for onehour at room temperature, in 2×SSC for 30 min at 50-60° C., and finallyin 0.1×SSC for 30 min at room temperature. Tissues were dehydrated ingraded ethanols and exposed to Kodak XAR-5 film for 3 days to 3 weeks at−20° C., then dipped in Kodak NTB3 autoradiography emulsion diluted 1:1with 0.2% glycerol water. After exposure at 4° C. for 2 to 8 weeks, theslides were developed in Kodak D-19 developer, fixed, and counterstainedwith cresyl violet.

Hybridization Controls

Controls for probe/hybridization specificity included hybridization withthe radiolabeled sense probe, and the use of transfected cell lines.Briefly, COS-7 cells were transfected (see above) with receptor cDNAsfor the rat Y1, Y2 (disclosed in U.S. patent application Ser. No.08/192,288, filed Feb. 3, 1994), Y4 (disclosed in U.S. patentapplication Ser. No. 08/176,412, filed Dec. 28, 1993), or Y5. Asdescribed above, the transfected cells were treated and hybridized withthe radiolabeled Y5 antisense and sense oligonucleotide probes, washed,and exposed to film for 1-7 days.

Analysis of Hybridization Signals

Sections through the rat brain were analyzed for hybridization signalsin the following manner. “Hybridization signal” as used in the presentcontext indicates the relative number of silver grains observed overneurons in a selected area of the rat brain. Two independent observersrated the intensity of the hybridization signal in a given brain area asnonexistent, low, moderate, or high. These were then converted to asubjective numerical scale as 0, +1, +2, or +3 (see Table 10), andmapped on to schematic diagrams of coronal sections through the ratbrain (see FIG. 11).

Chemical Synthetic Methods

Compounds evaluated in the in vitro Y5 receptor binding and functionalassays, and in vivo feeding assays of the present invention weresynthesized according to the methods described below.

It is generally preferred that the respective product of each processstep, as described hereinbelow, is separated and/or isolated prior toits use as starting material for subsequent steps. Separation andisolation can be effect by any suitable purifiaction procedure such as,for example, evaporation, crystallization, column chromatography, thinlayer chromatography, distillation, etc. While preferred reactants havebeen identified herein, it is further contemplated that the presentinvention would include chemical equivalents to each reactantspecifically enumerated in this disclosure. Temperatures are given indegrees Centigrade (°C.). The structure of final products, intermediatesand starting materials is confirmed by standard analytical methods,e.g., microanalysis and spectroscopic characteristics (e.g. MS, IR,NMR). Unless otherwise specified, chromatography is carried out usingsilica gel. Flash chromatography refers to medium pressure columnchromatography according to Still et al., J. Org. Chem. 43, 2928 (1978).

Synthesis of Compounds 1, 2, 5, 6, 7, 9, 10, and 11

For Compounds 1, 2, 5, 6, 7, 9, 10, and 11, thin layer chromatographywas performed using the following solvent system: A1:dichloromethane/methanol 9:1 A2: dichloromethane/methanol 19:1  A3:dichloromethane/methanol/ammonium hydroxide 90:10:1 B1:toluene/ethylacetate 1:1 B2: toluene/ethylacetate 10:1  C1:hexanes/ethylacetate 4:1 C2: hexanes/ethylacetate 3:1 C3:hexanes/ethylacetate 2:1

Compound 1: 2.4-Diphenylamino-quinazoline hydrochloride

2-Chloro-4-phenylamino-quinazoline (7.671 g) and aniline (3.627 g) areheated for 3 min to produce a melt which is dissolved in methanol. Theproduct is obtained as its hydrochloride salt upon addition of a slightexcess of 4N HCl in dioxane. Recrystallization from isopropanol yields2,4-diphenylamino-quinazoline hydrochloride, m.p. 319-320° C., FAB-MS(Fast Atom Bombardment Mass Spectroscopy): (M+H)⁺=313. Analytical data:C₂₀H₁₆N₄+HCl+0.5 H₂O, m.p. 319-320° C.

The starting material can be prepared as follows:

a) 2-Chloro-4-phenylamino-quinazoline

A solution of 2,4-dichloro-quinazoline (15 g),N,N-diisopropyl-ethylamine (24.9 ml) and aniline (7.5 ml) in isopropanol(75 ml) is heated to reflux for 45 min. The cold reaction mixture isfiltered and the filtrate is concentrated in vacuo. The residue iscrystallized from diethylether-toluene (1:1) to give2-chloro-4-phenyl-amino-quinazoline, m.p. 194-196° C.

b) 2,4-Dichloro-quinazoline

N,N-Dimethylaniline (114.0 g) is added slowly to a solution of1H,3H-quinazolin-2,4-dione (146.0 g) in phosphorousoxychloride (535.4ml) while this mixture is heated up to 140° C. After completion of theaddition reflux is continued for 20 h. The reaction mixture is filteredand evaporated to give a residue which is added to ice and water. Theproduct is extracted with dichloromethane and crystallized fromdiethylether and petroleum diethylether to yield2,4-dichloro-quinazoline, m.p. 115-116° C.

Compound 2: Naphthalene-1-sulfonic acid[6-(4-amino-quinazolin-2-ylamino)-hexyl]-amide

A solution of naphthalene-1-sulfonic acid (6-amino-hexyl)-amide (0.450g) and 2-chloro-quinazolin-4-ylamine (see: U.S. Pat. No. 3,956,495)(0.264 g) in 20 ml of isopentylalcohol is heated up to 120° C. for 15 h.Concentration of the reaction mixture followed by chromatography onsilica gel (B1) yields naphthalene-1-sulfonic acid[6-(4-amino-quinazolin-2-ylamino)-hexyl]-amide as a white powder,melting at 98-101° C. Rf(B1) 0.28, FAB-MS: (M+H)⁺=450. AnanlyticalC₂₆H₂₉N₅O₂S+HCl+H₂O+0.6 1,4 dioxane. m.p. 98-101° C.

Compound 5: trans-Naphthalene-1-sulfonic acid{4-[(4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethyl}-amidehydrochloride

A suspension of 2-chloro-quinazolin-4-ylamine (7.02 g) andtrans-naphthalene-1-sulfonic acid (4-aminomethyl-cyclohexylmethyl)-amide(13 g) in 250 ml of isopentyl-alcohol is heated up to 120° C. for 15 h.The resulting solution is concentrated and chromatographed (silica gel,B2) to give the product as a foam. This material is taken up indichloromethane (250 ml) and treated at 0° C. with a 4 N HCl solution indioxane (10 ml). Concentration in vacuo provides a foam which istriturated in boiling cyclohexane to yield after filtrationtrans-naphthalene-1-sulfonic acid{4-[(4-amino-quinazolin-2-ylamino)-methyl]-cyclohexylmethyl}-amidehydrochloride melting at 155-164° C. Rf(B2) 0.23, FAB-MS: (M+H)⁺=476.m.p. 155-164° C.

The starting material is prepared as follows:

a) trans-(4-Hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butylester

A solution oftrans-4-(tert-butoxycarbonylamino-methyl)-cyclohexanecarboxylic acid(obtained according to: EP 0614 911 A1) (34.5 g) and triethylamine (28ml) in dichloromethane (700 ml) is cooled to −70° C. and treated withmethylchloroformate (12.9 ml). The reaction mixture is stirred 0.5 h at−70° C. The temperature is allowed to increase to 0° C. and the solutionis stirred another 0.5 h until completion of the reaction. The reactionmixture is taken up in ice-cold dichloromethane, washed with an ice-cold0.5 N HCl solution, a saturated aqueous sodium carbonate solution andwater. The organics are dried over sodium sulfate and concentrated to41.3 g of mixt-anhydride as an oil. This material is taken up in THF andtreated at −70° C. with sodium borohydride (5.90 g), followed byabsolute methanol (10 ml). The reaction mixture is stirred 15 h at 0° C.and 1 h at ambient temperature to drive the reaction to completion. A0.5 N HCl solution is then carefuly added at 0° C., followed by ethylacetate. The organics are washed with a saturated aqueous sodiumcarbonate solution, water, dried over sodium sulfate and concentrated.Chromatography on silica gel (Al) yieldstrans-(4-hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butyl esteras a white powder, melting at 88-89° C. Rf(A1) 0.24.

b) trans-(4-Azidomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester

trans-(4-Hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester(24 g) in pyridine (200 ml) at 0° C. is treated with a solution ofpara-toluenesulfonylchloride (24.44 g) in pyridine (50 ml). The reactionmixture is stirred at 0° C. until completion and concentrated in vacuo.The residue is taken up in ethyl acetate, washed with water and driedover sodium sulfate. Concentration of the solution yields the tosylate,used without further purification. This material is treated with sodiumazide (19.23 g) in N,N-dimethylformamide (800 ml) at 50° C. Aftercompletion of the reaction, the solution is concentrated and theresulting paste is taken up in dichloromethane, washed with water andconcentrated. Chromatography of the crude material on silica gel (A2then A3) provides trans-(4-azidomethyl-cyclohexylmethyl)-carbamic acidtert-butyl ester as an oil. Rf(A3) 0.33; IR (dichloromethane) λ max 2099cm⁻¹.

c) trans-(4-Aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester

trans-(4-Azidomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester(24 g) in ethyl acetate (1 liter) is hydrogenated over platinumoxide(2.4 g) at ambient temperature under atmospheric pressure of hydrogen.The catalyst is filtered-off and the filtrate concentrated to yieldtrans-(4-aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester asan oil. Rf(C2) 0.41.

d)trans-{4-[(Naphthalene-1-sulfonylamino)-methyl]-cyclohexylmethyl}-carbamicacid tert-butyl ester

A solution of trans-(4-aminomethyl-cyclohexylmethyl)-carbamic acidtert-butyl ester (17 g) and ethyldiisopropylamine (14.41 ml) inN,N-dimethylformamide (350 ml) is cooled to 0° C. and treated with asolution of naphthalene-1-sulfonylchloride (15.9 g) inN,N-dimethylformamide (100 ml). The reaction is stirred at ambienttemperature for 2 h, concentrated in vacuo. The residue is taken up indichloromethane, washed with a 0.5 N HCl solution, a saturated aqueoussodium carbonate solution and water, dried and concentrated.Crystallization from hexanes-ethyl acetate givestrans-{(4-[<naphthalene-1-sulfonylamino)-methyl]-cyclohexylmethyl}-carbamicacid tert-butyl ester as a white powder, melting at 199-200° C. Rf(Al)0.42.

e) trans-Naphthalene-1-sulfonic acid(4-aminomethyl-cyclohexylmethyl)-amide

A suspension oftrans-{4-[(naphthalene-1-sulfonylamino)-methyl]-cyclohexylmethyl}-carbamicacid tert-butyl ester (25 g) in chloroform (300 ml) is treated with a 4N HCl solution in dioxane (300 ml) at 0° C. After completion, thereaction mixture is concentrated in vacuo, the residue is taken up in a1 N sodium hydroxide solution and dichloromethane. After extraction withdichloromethane, the organics are dried over sodium sulfate andconcentrated to 18.5 g of trans-naphthalene-1-sulfonic acid(4-aminomethyl-cyclohexylmethyl)-amide as a white powder melting at157-162° C. Rf(C3) 0.36.

Compound 6: 2-[4-(Piperidin-1-yl)-phenylamino]-4-phenylamino-quinazolinedihydrochloride

A mixture of 2-chloro-4-phenylamino-quinazoline (0.18 g) andN-(4-aminophenyl)-piperidine (0.164 g) is heated for 3 min to produce amelt which is dissolved in isopropanol (4 ml). 4 N HCl in dioxane (1 ml)is added. Recrystallization from ethanol and diethylether yields2-[4-(piperidin-1-yl)-phenylamino]-4-phenylamino-quinazolinedihydrochloride, Rf (Al) 0.64, FAB-MS: (M+H)⁺=396. m.p.:(decomposition).

Compound 7:trans-2-(4-Acetoxy-cyclohexylamino)-4-phenylamino-quinazolinehydrochloride

A solution oftrans-2-(4-hydroxy-cyclohexyamino)-4-phenylamino-quinazolinehydrochloride (1.3 g) and acetic anhydride (0.33 ml) in acetic acid (5ml) is stirred at ambient temperature for 16 h. The solvent is removedin vacuo and the residue is added to 2N aqueous NaOH. Extraction withethyl acetate followed by chromatography on silica gel (A4) gives acrude product which is treated with 4 N HCl in dioxane. Crystallizationfrom acetonitrile and acetone yieldstrans-2-(4-acetoxy-cyclohexylamino)-4-phenylamino-quinazolinehydrochloride, m.p. 217-220° C.; FAB-MS: (M+H)⁺=377; analytical data:C₂₂H₂₄N₄O₂+HCl.

The starting material is prepared as follows:

a) 2-(4-Hydroxy-cyclohexyamino)-4-phenylamino-quinazoline hydrochloride

A mixture of 2-chloro-4-phenylamino-quinazoline (2.3 g) andtrans-4-amino-cyclohexanol (1.26 g) is heated for 3 min to produce amelt which is dissolved in isopropanol.

4 N HCl in dioxane (0.1 ml) is added. Crystallization from isopropanoland acetone yields2-(4-hydroxy-cyclohexyamino)-4-phenylamino-quinazoline hydrochloride,m.p. 258-259° C.

Compound 9:8-Methoxy-2-(4-methoxy-phenylamino)-4-phenylamino-quinazolinehydrochloride

A mixture of 2-chloro-8-methoxy-4-phenylamino-quinazoline (1.20 g) and4-methoxy-aniline (0.66 g) is heated for 3 min to produce a melt whichis dissolved in isopropanol (15 ml). 4N HCl in dioxane (0.2 ml) isadded. Crystallization from isopropanol and diethylether yields8-methoxy-2-(4-methoxy-phenylamino)-4-phenylamino-quinazolinedihydrochloride, m.p. 287-289° C., FAB-MS: (M+H)⁺=373. Analytical data:C₂₂H₂₀N₄O₂+HCl.

The starting material can be prepared as follows:

a) 2-Chloro-8-methoxy-4-phenylamino-quinazoline

A solution of 2,4-dichloro-8-methoxy-quinazoline (prepared as describedin J. Chem. Soc. 1948, 1759) (0.6 g), N,N-diisopropyl-ethylamine (0.87ml), and aniline (0.26 ml) in isopropanol (10 ml) is heated to refluxfor 45 min. The cold reaction mixture is filtered and residue iscrystallized from dichloromethane and hexanes to give2-chloro-8-methoxy-4-phenylamino-quinazoline, m.p. 245-246° C.

Compound 10:N-Methyl-[4-(6-methoxy-4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride

A solution of 2-chloro-6-methoxy-4-phenylamino-quinazoline (1.15 g) andN-methyl-(4-aminophenyl)-methanesulfonamide (prepared as described inTetrahedron Letters 1992, 33, 8011) (0.89 g) in 5 ml of isopentylalcoholis stirred under nitrogen at 180° C. for 20 min in a sealed vessel. Thewarm reaction mixture is diluted with methanol and the hydrochloridesalt, which is crystallizing on cooling, is filtered off. The crudeproduct is redissolved in ethylacetate and aqueous sodium carbonatesolution and extracted with ethylacetate. The organic extracts are driedand evaporated and the solid residue is titurated with diethylether togiveN-methyl-[4-(6-methoxy-4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamideas light yellow crystals melting at 212-215° C.; (Rf (A2) 0.16.Recrystallisation from methanolic hydrogen chloride and diethyletheryieldsN-methyl-[4-(6-methoxy-4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride as light yellow crystals melting at 264-268° C.; Rf (A2)0.16, FAB-MS: (M+H)⁺=450. Analytical data: C₂₃H₂₃N₅O₃S+HCl.

The starting material can be prepared as follows:

a) 2-Chloro-6-methoxy-4-phenylamino-quinazoline

In a procedure analogous to that of Example 1a2,4-dichloro-6-methoxy-quinazoline (1.53 g) (prepared as described in J.Chem. Soc. 1948, 1759), aniline (0.8 g) (0.184 g) andN,N-diisopropyl-ethylamine (1.72 g) are reacted together to give2-chloro-6-methoxy-4-phenylamino-quinazoline as light yellow crystalsmelting at 177-179° C., Rf (A2) 0.59.

Compound 11:N-Methyl-[4-(4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride

A solution of 2-chloro-4-phenylamino-quinazoline (0.92 g) (prepared asdescribed in Example 1a and N-methyl-(4-aminophenyl)-methanesulfonamide(0.80 g) in 10 ml of isopentylalcohol is stirred under nitrogen at 170°C. for 15 min in a sealed vessel. The warm reaction mixture is dilutedwith 10 ml ethanol and the hydrochloride salt, which is crystallizing oncooling, is filtered off to yieldN-methyl-[4-(4-phenylamino-quinazolin-2-ylamino)-phenyl]-methanesulfonamidehydrochloride as light yellow crystals melting at 259-263° C.; Rf (A2)0.11, FAB-MS: (M+H)⁺=420. Analytical data: C₂₂H₂₁N₅O₂S+HCl.

Synthesis of Compounds 17-23, Compound 26 and Compound 27

Compounds 17-23, 26 and 27 were synthesized according to the generalmethod in Scheme 1, as described below. An example of the synthesis of aspecific compound, Compound 17, follows the general description.Compounds 18-23, 26 and 27 were synthesized in the same manner but usingthe appropriately substituted starting materials.

Preparation of the compounds of the present invention having thestructure shown in Formula 1-3, Scheme 1, is carried out usingwell-known methodology for the preparation of a sulfonamide from anamine. Preferably the appropriate arylsulfonyl halide, preferably thechloride (i.e., Ar—SO₂Cl), is reacted with a monoprotected linear orcyclic alkylamine (Krapcho and Kuell, Synth. Comm. 20(16): 2559-2564,1990) comprising H₂N-L-K″, where K″ comprises methylene, in the presenceof a base such as a tertiary amine, e.g., triethylamine,dimethylaminopyridine, pyridine or the like, in an appropriate solvent(e.g. CHCl₃, CH₂Cl₂) as shown in Scheme 1, step A, followed bydeprotection of the resulting amine as shown in Scheme 1, Step B, allunder mild conditions (typically room temperature), to yield thedeprotected amine of Formula 1-1. The arylsulfonyl halides are eitherknown in the art or can be prepared according to methods well known inthe art. Compounds of Formula 1-2 in Scheme 1, may be synthesized fromthe compound of Formula 1-1 by amidation using suitable methods such asthose taught in “The Peptides,” Vol. 1 (Gross and Meinehofer, Eds.Acaemic Press, N.Y., 1979). For example, the compound of Formula 1-1 maybe treated with a carboxylic acid derivative of W in the presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) anddimethylaminopyridine (DMAP) in a suitable solvent such as CH₂Cl₂ asshown in Scheme 1, Step C, at room temperature in an inert atmosphere ofargon or nitrogen, to yield the amide compound of Formula 1-2. The K″amine and the carboxylic acid carbon attached to W together form K inthe product.

Alternatively, the compound of Formula 1-2 may be synthesized byacylation of the amine of Formula 1-1 using the acid chloride of W,i.e., WCOCl, in a solvent such as CH₂Cl₂ and a suitable tertiary aminesuch as triethylamine, at room temperature. Again, the K″ amine and theacid chloride carbon attached to W together form K in the product.

The product compounds of Formula 1-3 are then formed by reduction of theamide of Formula 1-3 using borane-tetrahydorfuran (THF) complex, in THFas shown in Scheme 1, Step D, at elevated temperature in an inertatmosphere.

As a specific example of the synthesis of compounds 17-23, 26 and 27,the synthesis of Compound 17 is given hereinbelow.

Compound 17: Naphthalene-2-sulfonic acid(4-[}(1, 2, 3,4-tetrahydronaphthalen-2-yl)methyl}-amino]-trans-yclohexylmethyl)-amide

Step A Scheme 1

{4-[(Naphthalene-2-sulfonylamino)-trans-cyclohexylmethyl]-carbamic acidtert-butyl ester

To a stirred solution of (4-aminomethyl-cyclohexylmethyl]-carbamic acidtert-butyl ester(0.50 g, 2.1 mmol) and triethyl amine ( 0.42 g, 4.2mmol) in 50 mL methylene chloride was added 2-naphthalenesulfonylchloride (0.51 g, 2.3 mmol). The reaction mixture was stirred for 6 h atroom temperature, quenched with brine, and extracted with methylenechloride(2×50 mL). The organic layer was washed with brine, dried overanhydrous sodium sulfate, and concentrated in vacuo to yield the titledcompound as white solid(0.74 g, 83%): mp 114-5° C.

Step B, Scheme 1

Naphthalene-2-sulfonic acid-(4-aminomethyl-trans-cyclohexylmethyl)-amide

To a stirred solution of {4-[(naphthalene-2-sulfonylamino)-transcyclohexylmethyl]-carbamic acid tert-butyl ester(0.73 g, 1.6 mmol) in 25mL of methylene chloride at room temperature was added 3 mL of saturatedHCl solution in ethyl acetate and stirred for 4 h. The precipitatedsolid was filtered to yield the titled compound as white solid (0.58 g,99%); mp 286-7° C,

Step C, Scheme 1

1, 2, 3, 4-Tetrahydronaphthalene-2-carboxylicacid[4-{(naphthalen-2-sulfonylamino)methyl}-tans-cyclohexylmethyl]amide

A mixture of naphthalene-2-sulfonicacid-(4-aminomethyl-trans-cyclohexylmethyl)amide (0.5 g, 1.4 mmol), EDC(0.54 g, 2.8 mmol), and DMAP (0.34 g, 2.8 mmol) in methylene chloride(30mL) was stirred at room temperature for 0.5 h.1,2,3,4-tetrahydronaphthalen-2-carboxylic acid (0.24 g, 1.4 mmol) wasadded to the reaction mixture and stirred at room temperature till thecompletion of the reaction(by TLC). The reaction mixture was washed withsaturated ammonium chloride (3×30 mL), dried over sodium sulfate andconcentrated in vacuo. The residue was flash chromatographed over silicagel to afford white solid (0.66 g, 99%); mp 225-6° C.

Step D, Scheme 1

Naphthalene-2-sulfonic acid(4-[{(1, 2, 3,4-tetrahydronaphthalen-2-yl)methyl}-amino]-trans-cyclohexylmethyl)-amide

To a solution of 1, 2, 3, 4-tetrahydronaphthalen-2-carboxylic acid[4-{(naphthalen-2-sulfonylamino)methyl}-transcycohexylmethyl]amide (0.65 g,1.3 mmol) in tetrahydrofuran(5 mL) cooled to 0° C. was added 6.6 mL 1Msolution of borane:THF complex and the reaction mixture was refluxed for12 h. The reaction mixture was cooled in ice bath and quenched with 2 mLof 1N HCl. The reaction mixture was neutralized with 10% aqueous sodiumhydroxide solution and extracted with ethyl acetate (3×25 mL). organicphase was washed with the brine, dried over sodium sulfate, evaporatedin vacuo to afford an oil which was purified by preparative TLC toafford the titled compound(0.44 g,70%); hydrochloride salt mp (210° C.).

In order to synthesize compounds 18-24, 26 and 27, the2-naphthalenesulfonyl chloride of Step A above, which comprises the “Ar”moiety of Table 2, is replaced with the appropriate Ar- sulfonylchloride, and the 1,2,3,4-tetrahydronaphthalen-2-carboxylic acid used inStep C above, which comprises the “W” moiety of Table 2, is replacedwith the appropriate W-carboxylic acid, to yield product containing thecorresponding Ar and W moieties shown in Table 2.

Synthesis of Compound 25

Compound 25 was synthesized according to Scheme 2. After protection ofH₂N-L-COOH with Boc anhydride in CH₂Cl₂, as shown in Scheme 2, Step A,the protected amine may be amidated with W—K′″ as in Scheme 2, Step B,where K′″ is (CH₂)jCHR₇—NH₂, where R₇ is an ester and j is 1 using EDCand DMAP in a suitable solvent such as CH₂Cl₂, to yield compounds ofFormula 3-1, where K′″ and the carboxylic acid carbonyl of H₂N-L-COOHtogether form K. The compounds of Formula 3-1 may be deprotected usingwell known methods as shown in Scheme 2, Step C, and furthersulfonylated with a sulfonyl halide of Ar, as shown in Scheme 2, Step D,in a suitable solvent such as CH₂Cl₂ and a tertiary amine such astriethylamine, to form the compound of Formula 3-3. Compounds of Formula3-3 may be reduced to yield the compounds of Formula 3-3, as shown inScheme 2, Step E, using borane-tetrahydorfuran (THF) complex, in THF, atelevated temperature in an inert atmosphere.

A detailed description of the synthesis of Compound 25 is given below:

Compound 25: trans-3- (4-Chloro-phenyl) -2-({[4-(naphthalene-1-sulfonylamino)-methyl]-cyclohexanecarbonyl}-amino]-propionicacid methyl ester

(a) Step A, Scheme 2

trans-4- (tert-Butoxycarbonylamino-methyl)-cyclohexanecarboxylic acid

To a solution of trans-4-(aminomethyl)cyclohexanecarboxylic acid (10 g,57 mmol) in 1 N NaOH (110 mL) cooled to 0° C. was added a solution ofdi-tert-butyl dicarbonate (15 g, 69 mmol) in dioxane (50 mL). Thereaction mixture was stirred at 0° C. for 12 h. The reaction mixture wasneutralized by 1 N HCl solution to pH 3, extracted with ethyl ether(2×300 mL), washed with brine (2×300 mL), dried over anhydrous magnesiumsulfate, and concentrated in vacuo to afford the titled compound (16 g,100%); white solid, mp 128-9° C.

(b) Step B, Scheme 2

trans-2-{([4-(tert-Butoxycarbonylamino-methyl)-cyclohexanecarbonyl]-amino}3-(4-Chloro-phenyl)-propionicacid methyl ester

Using the general procedure described for the preparation Step B, Scheme2, trans-4-(tert-butoxycarbonylamino-methyl)-cyclohexanecarboxylic acid(1.1 g, 4.0 mmol) was acylated with D,L-4-chlorophenylalanine methylester hydrochloride (1.0 g, 4.0 mmol) to afford the titled compound (1.9g, 99%); white solid, mp 178-9° C.

(c) Step C, Scheme 2

trans-2-[4-(Aminomethyl-cyclohexanecarbonyl)-amino]3-(4-chloro-phenyl)-propionicacid methyl ester hydrochloride

Using the general procedure described in step C Scheme 2,trans-2-{[4-(tert-butoxycarbonylamino-methyl)-cyclohexanecarbonyl]-amino}3-(4-chloro-phenyl) -propionic acid methyl ester (1.8 g, 4.3 mmol) wasdeprotected using HCl in ethyl acetate to afford the titled compound;light yellow solid mp 146-9° C.

(d) Step D, Scheme 2

trans-3-(4-Chloro-phenyl)-2-({[4-(naphthalene-1-sulfonylamino)-methyl]-cyclohexanecarbonyl}-amino]-propionicacid methyl ester:

Using the general procedure described in step B Scheme 2,trans-2-[4-(aminomethyl-cyclohexanecarbonyl)-amino]3-(4-Chloro-phenyl)-propionicacid methyl ester hydrochloride (0.35 g, 0.86 mmol) was sulfonylatedwith 1-naphthalenesulfonyl chloride (0.42 g, 91%) to afford the titledcompound; white solid, mp 84-6° C.

Compound 25 was synthesized from the above compound by borane-THFreduction as follows:

(e) Step E, Scheme 2

Naphthalene-1-sulfonic Acid trans-(4-{[2-(4-Chloro-phenyl)-1-hydroxymethyl-ethylamino]-methyl}-cyclohexylmethyl)-amide

Using the general procedure described in Step E, Scheme 2,trans-3-(4-chloro-phenyl)-2-({[4-(naphthalene-1-sulfonylamino)-methyl]-cyclohexanecarbonyl}-amino]-propionicacid methyl ester (0.30 g, 0.55 mmol) was reduced by borane:THF complex(1.0 M in THF) to afford the titled compound; colorless oil.

Synthesis of Compound 28

2-(Naphthalen-1-ylamino)-3 -phenylpropionitrile

To a solution of 1-naphthalenemethylamine (2.9 g, 20 mmol) andbenzylaldehyde (2.0 g, 17 mmol) in 30 ml of CHCl₃ and 10 ml of MeOH wasadded TMSCN (6.6 ml, 51 mmol) and the resulting solution was stirred for12 h at 25° C. The reaction mixture was concentrated in vacuo, yieldingan oil which was subjected to column chromatography (EtOAc, neat) toprovide 3.5 g (74%) of the desired product as a colorless oil. Productwas identified by NMR.

2- (Naphthalen-1-yl) -3-phenylpropane-1,2-diamine

To a solution of the nitrile (0.5 g, 1.8 mmol) in THF was added 6.9 mlof 1N LiAlH₄ in THF dropwise and the resulting solution was stirred for2 h. The reaction was quenched by adding a few pieces of ice into thesolution. The reaction mixture was diluted with EtOAc and filteredthrough pad of Celite. Organic filtrate was concentrated in vacuo toprovide a oily residue which was subjected to column chromatography(EtOAc, neat) to provide 0.28 g (57%) of the desired product as acolorless oil. The product was identified by NMR. TABLE 2 No. Ar X R₁ LK W mp Analysis 17

— H

CH₂NHCH₂

210 C₂₉H₃₆N₂O₂S +HCl 19

— H

CH₂NHCH₂

220 C₂₉H₃₆N₂O₂S +HCl + 0.15 CH₂Cl₂ 20

— H

CH₂NHCH₂

200-2 C₂₅H₃₃N₃O₄S +HCl 21

— H

CH₂NHCH₂

171-4 C₂₆H₂₉N₂O₂SF₃ +HCl + 0.075 CHCl₃ 22

— H

CH₂NHCH₂

175-7 C₂₅H₃₅N₃O₂S +2 HCl + 0.8 Et₂O 23

— H

CH₂NHCH₂

216-7 C₂₆H₂₉N₂O₂SF₃ +HCl 25

— H

223-3 C₂₇H₃₃N₂O₂SCl +HCl 26

— H

CH₂NHCH₂

89 dec C₂₄H₂₈N₄O₄S +HCl 27

— H

CH₂NHCH₂

104-6 C₂₅H₂₈N₄O₄S +2 HCl + 0.2 CHCl₃In vivo STUDIES IN RATSFood Intake in Satiated Rats

For these determinations food intake may be measured in normal satiatedrats after intracerebroventricular application (i.c.v.) of NPY in thepresence or absence of the test compound. Male Sprague Dawley rats(Ciba-Geigy A G, Sisseln, Switzerland) weighing between 180 g and 220 gare used for all experiments. The rats are individually housed instainless steel cages and maintained on an 11:13 h light-dark cycle(lights off at 18:00 h) at a controlled temperature of 21-23° C. at alltimes. Water and food (NAFAG lab chow pellets NAFAG, Gossau,Switzerland) are available ad libidum.

Rats under pentobarbital anesthesia are stereotaxically implanted with astainless steel guide cannula targeted at the right lateral ventricle.Stereotaxic coordinates, with the incisor bar set −2.0 mm belowinteraural line, are: −0.8 mm anterior and +1.3 mm lateral to bregma.The guide cannula is placed on the dura. Injection cannulas extend theguide cannulas −3.8 mm ventrally to the skull surface. Animals areallowed at least 4 days of recovery postoperatively before being used inthe experiments. Cannula placement is checked postoperatively by testingall rats for their drinking response to a 50 ng intracerebroventricular(i.c.v.) injection of angiotensin II. Only rats which drink at least 2.5ml of water within 30 min. after angiotensin II injection are used inthe feeding studies.

All injections are made in the morning 2 hours after light onset.Peptides are injected in artificial cerebrospinal fluid (ACSF) in avolume of 5 μl. ACSF contains: NaCl 124 mM, KCl 3.75 mM, CaCl₂ 2.5 mM,MgSO₄ 2.0 mM, KH₂PO₄ 0.22 mM, NaHCO₃ 26 mM and glucose 10 mM.Porcine-NPY (p-NPY) are dissolved in artificial cerebrospinal fluid(ACS). For i.c.v. injection the test compounds are preferably dissolvedin DMSO/water (10%, v/v). The vehicle used for intraperitoneal (i.p.) ,subcutaneous (s.c.) or oral (p.o.) delivery of compounds is preferablywater, physiological saline or DMSO/water (10% v/v), or cremophor/water(20% v/v), respectively.

Animals which are treated with both test compounds and porcine-NPY aretreated first with the test compound. Then, 10 min. after i.c.v.application of the test compound or vehicle (control), or for i.p.,s.c., or p.o. -administration, 30-60 min after application of the testcompound or vehicle, generally, NPY is administered byintracerebroventricular (i.c.v.) application.

Food intake may be measured by placing preweighed pellets into the cagesat the time of NPY injection. Pellets are then removed from the cagesubsequently at each selected time point and replaced with a new set ofpreweighed pellets. The food intake of animals treated with testcompound may be calculated as a percentage of the food intake of controlanimals i.e., animals treated with vehicle. Alternatively, food intakefor each group of animals subjected to a particular experimentalcondition may be expressed as the mean+S.E.M. Statistical analysis isperformed by analysis of variance using the Student-Newman-Keuls test.

Food Intake in Food-Deprived Rats

Food-deprivation experiments are conducted with male Sprague-Dawley ratsweighing between 220 and 250 g. After receipt, the animals areindividually housed for the duration of the study and allowed freeaccess to normal food together with tap water. The animals aremaintained in a room with a 12 h light/dark cycle (8:00 a.m. to 8:00p.m. light) at 24° C. and monitored humidity.

After placement into individual cages the rats undergo a 4 dayequilibration period, during which they are habituated to their newenvironment and to eating a powdered or pellet diet NAFAG, Gossau,Switzerland).

At the end of the equilibration period, food is removed from the animalsfor 24 hours starting at 8:00 a.m. At the end of the fasting periodcompound or vehicle may be administered to the animals orally or byinjection intraperitoneally or intravenously. After 10- 60 min. food isreturned to the animals and their food intake is monitored at varioustime periods during the following 24 hour period. The food intake ofanimals treated with test compound may be calculated as a percentage ofthe food intake of control animals (i.e., animals treated with vehicle).Alternatively, food intake for each group of animals subjected to aparticular experimental condition may be expressed as the mean+S.E.M.

Food Intake in Obese Zucker Rats

The antiobesity efficacy of the compounds according to the presentinvention might also be manifested in Zucker obese rats, which are knownin the art as an animal model of obesity. These studies are conductedwith male Zucker fatty rats (fa/fa Harlan CPB, Austerlitz NL) weighingbetween 480 g and 500 g. Animals are individually housed in metabolismcages for the duration of the study and allowed free access to normalpowdered food and water. The animals are maintained in a room with a 12h light/dark cycle (light from 8:00 A.M. to 8:00 P.M.) at 24° C. andmonitored humidity. After placement into the metabolism cages the ratsundergo a 6 day equilibration period, during which they are habituatedto their new environment and to eating a powdered diet. At the end ofthe equilibration period, food intake during the light and dark phasesis determined. After a 3 day control period, the animals are treatedwith test compounds or vehicle (preferably water or physiological salineor DMSO/water (10%, v/v) or cremophor/water (20%, v/v)). Food intake isthen monitored over the following 3 day period to determine the effectof administration of test compound or vehicle alone. As in the studiesdescribed hereinabove, food intake in the presence of drug may beexpressed as a percentage of the food intake of animals treated withvehicle, or as the amount of food intake for a group of animalssubjected to a particular experimental condition.

Materials

Cell culture media and supplements are from Specialty Media (Lavallette,NJ). Cell culture plates (150 mm and 96-well microtiter) were fromCorning (Corning, N.Y.). Sf9, Sf21, and High Five insect cells, as wellas the baculovirus transfer plasmid, pBlueBacIII™, were purchased fromInvitrogen (San Diego, Calif.). TMN-FH insect medium complemented with10% fetal calf serum, and the baculovirus DNA, BaculoGold™, was obtainedfrom Pharmingen (San Diego, Calif.). Ex-Cell 400™ medium withL-Glutamine was purchased from JRH Scientific. Polypropylene 96-wellmicrotiter plates were from Co-star (Cambridge, Mass.). All radioligandswere from New England Nuclear (Boston, Mass.). Commercially availableNPY and related peptide analogs were either from Bachem California(Torrance, Calif.) or Peninsula (Belmont, Calif.); [D-Trp³²]NPY and PPC-terminal fragments were synthesized by custom order from ChironMimotopes Peptide Systems (San Diego, Calif.). Bio-Rad Reagent was fromBio-Rad (Hercules, Calif.). Bovine serum albumin (ultra-fat free,A-7511) was from Sigma (St. Louis. Mo.). All other materials werereagent grade.

EXPERIMENTAL RESULTS

cDNA Cloning

In order to clone a rat hypothalamic “atypical” NPY receptor subtype,applicants used an expression cloning strategy in COS-7 cells (Gearinget al, 1989; Kluxen et al, 1992; Kiefer et al, 1992). This strategy waschosen for its extreme sensitivity since it allows detection of a single“receptor positive” cell by direct microscopic autoradiography. Sincethe “atypical” receptor has only been described in feeding behaviorstudies involving injection of NPY and NPY related ligands in rathypothalamus (see introduction), applicants first examined its bindingprofile by running competitive displacement studies of ¹²⁵I-PYY and¹²⁵I-PYY₃₋₃₆ on membranes prepared from rat hypothalamus. Thecompetitive displacement data indicate: 1) Human PP is able to displace20% of the bound ¹²⁵I-PYY with an IC₅₀ of 11 nM (FIG. 1 and Table 3). Ascan be seen in Table 5, this value does not fit with the isolated ratY1, Y2 and Y4 clones and could therefore correspond to another NPY/PYYreceptor subtype. 2) [Leu₃₁, Pro₃₄]NPY (a Y1 specific ligand) is able todisplace with high affinity (IC₅₀ of 0.38) 27% of the bound ¹²⁵I-PYY₃₋₃₆ligand (a Y2 specific ligand) (FIG. 2 and Table 3). These data providethe first evidence based on a binding assay that rat hypothalamicmembranes could carry an NPY receptor subtype with a mixed Y1/Y2pharmacology (referred to as the “atypical” subtype) which fits with thepharmacology defined in feeding behavior studies.

TABLE 3: Pharmacological Profile of the Rat Hypothalamus.

Binding data reflect competitive displacement of ¹²⁵I-PYY and ¹²⁵I-PYY₃₋₃₆ from rat hypothalamic membranes. Peptides were tested atconcentrations ranging from 0.001 nM to 100 nM unless noted. The IC₅₀value corresponding to 50% displacement, and the percentage ofdisplacement relative to that produced by 300 nM human NPY, weredetermined by nonlinear regression analysis. Data shown arerepresentative of at least two independent experiments. TABLE 3 IC₅₀Values, nM (% NPY- produced displacement) Peptide ¹²⁵I-PYY ¹²⁵I-PYY₃₋₃₆human NPY 0.82 (100%)  1.5 (100%) human NPY₂₋₃₆  2.3 (100%)  1.2 (100%)human 0.21 (44%) 0.38 (27%) [Leu³¹, Pro³⁴]NPY  340 (56%)  250 (73%)human PYY  1.3 (100%) 0.29 (100%) human PP   11 (20%) untested

Based on the above data, a rat hypothalamic cDNA library of 3×10⁶independent recombinants with a 2.7 kb average insert size wasfractionated into 450 pools of ˜7500 independent clones. All pools weretested in a binding assay with ¹²⁵I-PYY as previously described (U.S.Ser. No. 08/192,288). Seven pools gave rise to positive cells in thescreening assay (#'s 81, 92, 147, 246, 254, 290, 312). Since Y1, Y2, Y4and Y5 receptor subtypes (by PCR or binding analysis) are expressed inrat hypothalamus, applicants analyzed the DNA of positive pools by PCRwith rat Y1, Y2 and Y4 specific primers. Pools # 147, 246, 254 and 312turned out to contain cDNAs encoding a Y1 receptor; pool # 290 turnedout to contain cDNA encoding a Y2 receptor subtype; but pools # 81 and92 were negative by PCR analysis for Y1, Y2 and Y4 and therefore likelycontained a cDNA encoding a new rat hypothalamic NPY receptor (Y5).Pools # 81 and 92 later turned out to contain an identical NPY receptorcDNA. Pool 92 was subjected to sib selection as described (U.S. Ser. No.08/192,288) until a single clone was isolated (designated CG-18).

The isolated clone carries a 2.8 kb cDNA. This cDNA contains an openreading frame between nucleotides 779 and 2146 that encodes a 456 aminoacid protein. The long 5′ untranslated region could be involved in theregulation of translation efficiency or mRNA stability. The flankingsequence around the putative initiation codon does not conform to theKozak consensus sequence for optimal translation initiation (Kozak,1989, 1991). The hydrophobicity plot displayed seven hydrophobic,putative membrane spanning regions which makes the rat hypothalamic Y5receptor a member of the G-protein coupled superfamily. The nucleotideand deduced amino acid sequences are shown in FIGS. 3 and 4,respectively. Like most G-protein coupled receptors, the Y5 receptorcontains consensus sequences for N-linked glycosylation, in the aminoterminus (position 21 and 28) involved in the proper expression ofmembrane proteins (Kornfeld and Kornfeld, 1985). The Y5 receptor carriestwo highly conserved cysteine residues in the first two extracellularloops that are believed to form a disulfide bond stabilizing thefunctional protein structure (Probst et al, 1992). The Y5 receptor shows9 potential phosphorylation sites for protein kinase C in positions 204,217, 254, 273, 285, 301, 328, 336 and 409 and 2 cAMP- and cGMP-dependentprotein kinase phosphorylation sites in positions 298 and 370. It shouldbe noted that 8 of these 11 potential phosphorylation sites are locatedin the third intra-cellular loop, two in the second intra-cellular loop,and one in the carboxy terminus of the receptor and could therefore playa role in regulating functional characteristics of the Y5 receptor(Probst et al, 1992). In addition the rat Y5 receptor carries a leucinezipper motif in its first putative transmembrane domain (Landschulz etal, 1988). A tyrosine kinase phosphorylation site is found in the middleof the leucine zipper.

Localization studies (see below) show that the Y5 mRNA is present inseveral areas of the rat hippocampus. Assuming a comparable localizationin human brain, applicants screened a human hippocampal cDNA library (asdescribed in U.S. Ser. No. 8/192,288) with rat oligonucleotide primerswhich were shown to yield a DNA band of the expected size in a PCRreaction run on human hippocampal cDNA (C. Gerald, unpublished results).Using this PCR screening strategy (Gerald et al, 1994, submitted forpublication), three positive pools were identified. One of these poolswas analyzed further, and an isolated clone was purified by sibselection. The isolated clone (CG-19) turned out to contain a fulllength cDNA cloned in the correct orientation for functional expression(see below). The human Y5 nucleotide and deduced amino acid sequencesare shown in FIGS. 5 and 6, respectively. When compared to the rat Y5receptor the human sequence shows 84.1% nucleotide identity (FIGS. 7A to7E) and 87.2% amino acid identity (FIGS. 7F and 7G). The rat proteinsequence is one amino acid longer at the very end of both amino andcarboxy tails of the receptor when compared to the rat. The human 5-6loop is one amino acid longer than the rat and shows multiple nonconservative substitutions. Even though the 5-6 loops show significantchanges between the rat and human homologs, all of the protein motifsfound in the rat receptor are present in the human homolog. All putativetransmembrane domains and extra cellular loop regions are highlyconserved (FIGS. 7F and 7G). Therefore, both pharmacological profilesand functional characteristics of the rat and human Y5 receptor subtypehomologs may be expected to match closely.

When the human and rat Y5 receptor sequences were compared to other NPYreceptor subtypes or to other human G protein-coupled receptor subtypes,both overall and transmembrane domain identities were very low, showingthat the Y5 receptor genes are not closely related to any otherpreviously characterized cDNAs (Table 4). Even among the human NPYreceptor family, Y1, Y2, Y4 and Y5 members show unusually low levels ofamino acid identity (FIG. 8A through 8C). TABLE 4 Human Y5 transmembranedomains identity with other human NPY receptor subtypes and other humanG-protein coupled receptors Receptor subtype % TM identity Y-4 40 Y-2 42Y-1 42 MUSGIR 32 DroNPY 31 Beta-1 30 Endothelin-1 30 Dopamine D2 29Adenosine A2b 28 Subst K 28 Alpha-2A 27 5-HT1Dalpha 26 Alpha-1A 26 IL-826 5-HT2 25 Subst P 24Northern Blot Analysis

Using the rat Y5 probe, northern hybridizations reveal a strong signalat 2.7 kb and a weak band at 8 kb in rat whole brain. A weak signal isobserved at 2.7 kb in testis. No signal was seen in heart, spleen, lung,liver, skeletal muscle and kidney after a three day exposure (FIG. 16A).This is in good agreement with the 2.7 kb cDNA that we isolated byexpression cloning from rat hypothalamus and indicates that our cDNAclone is full length. The 8 kb band seen in whole brain probablycorresponds to unspliced pre-mRNA.

With the human Y5 probe, northern hybridizations (FIGS. 16B and 16C)showed a strong signal at 3.5 kb with a much weaker band at 2.2 and 1.1kb in caudate nucleus, putamen and cerebral cortex, a medium signal infrontal lobe and amygdala and a weak signal in hippocampus, occipitaland temporal lobes, spinal cord,. medulla, thalamus, subthalamicnucleus, and substantia nigra. No signal at 3.5 kb was detectable incerebellum or corpus callosum after a 48 h exposure. It should be notedthat Clontech's MTN II and III blots do not carry any mRNA fromhypothalamus, periaquiductalgray, superior colliculus and raphe.

Southern blot analysis on human genomic DNA reveals a unique bandpattern in 4 of the 5 restriction digests (FIG. 17A). The two bandsobserved in the PstI digest can be explained by the presence of a PstIsite in the coding region of the human Y5 gene. Rat southern blottinganalysis showed a unique band pattern in all five restriction digeststested (FIG. 17B). These analyses are consistent with the human and ratgenomes containing a single copy of the Y5 receptor gene.

Canine Y5 Homolog

The canine nucleotide sequence obtained to date (PCR and 3′ RACEproducts) spans the canine Y5 receptor from the first extracellular loopimmediately upstream of TM III into the 3′ untranslated region (FIG.14). In the coding region, this nucleotide sequence is highly identicalto both the human and the rat sequences (91% and 83.3% respectively).The deduced canine Y5 amino acid sequence is shown in FIG. 15. Thisamino acid sequence is again highly identical to both the human and ratY5 sequences (94.6% and 89.5% respectively), with most amino acidchanges located in the 5-6 loop. Therefore the pharmacological profileof the canine Y5 receptor subtype is expected to closely resemble thehuman and rat Y5 profiles.

Binding Studies

The cDNA for the rat hypothalamic Y5 receptor was transiently expressedin COS-7 cells for full pharmacological evaluation. ¹²⁵I-PYY boundspecifically to membranes from COS-7 cells transiently transfected withthe rat Y5 receptor construct. The time course of specific binding wasmeasured in the presence of 0.08 nM ¹²⁵I-PYY at 30° C. (FIG. 9). Theassociation curve was monophasic, with an observed association rate(K_(obs)) of 0.06 min⁻¹ and a t_(1/2) of 11 min; equilibrium binding was99% complete within 71 min and stable for at least 180 min. Allsubsequent binding assays were carried out for 120 min at 30° C. Thebinding of ¹²⁵I-PYY to transiently expressed rat Y5 receptors wassaturable over a radioligand concentration range of 0.4 pM to 2.7 nM.Binding data were fit to a one-site binding model with an apparent K_(d)of 0.29 nM (pK_(d)=9.54±0.13, n=4). A receptor density of between 5 and10 pmol/mg membrane protein was measured on membranes which had beenfrozen and stored in liquid nitrogen (FIG. 10). Membranes frommock-transfected cells, when prepared and analyzed in the same way asthose from CG-18-transfected cells, displayed no specific binding of¹²⁵I-PYY (data not shown). Applicants conclude that the ¹²⁵I-PYY bindingsites observed under the described conditions were derived from the ratY5 receptor construct.

A closely related peptide analog, porcine ¹²⁵I-[Leu³¹,Pro³⁴]PYY, alsobound specifically to membranes from COS-7 cells transiently transfectedwith rat Y5 receptor cDNA. The time course of specific binding wasmeasured at room temperature in both standard binding buffer ([Na⁺]=10mM) and isotonic binding buffer ([Na⁺]=138 mM) using 0.08 nM nM¹²⁵I-[Leu³¹,Pro³⁴]PYY nM (FIG. 18). The association curve in 10 mM [Na⁺]was monophasic, with an observed association rate (K_(obs)) of 0.042min⁻¹ and a t_(1/2) of 17 min; equilibrium binding was 99% completewithin 110 min and stable for at least 210 min (specific binding wasmaximal at 480 fmol/mg membrane protein). The association curve in 138mM [Na⁺] was also monophasic with a slightly slower time course:(K_(obs)) of 0.029 min⁻¹ and a t_(1/2) of 24 min.; equilibrium bindingwas 99% complete within 160 min. and stable for at least 210 min.(specific binding was maximal at 330 fmol/mg membrane protein). Notethat the specific binding was reduced as [Na⁺] was increased; a similarphenomenon has been observed for other G protein coupled receptors andmay reflect a general property of this receptor family to be modulatedby Na⁺ (Horstman et. al., 1990). Saturation binding studies wereperformed with ¹²⁵I-[Leu³¹,Pro³⁴]PYY in isotonic buffer at roomtemperature over a 120 minute period. Specific binding to transientlyexpressed rat Y5 receptors was saturable over a radioligandconcentration range of 0.6 pM to 1.9 nM. Binding data were fit to aone-site binding model with an apparent K_(d) of 0.072 nM(pKd=10.14+0.07, n=2). A receptor density of 560±150 pmol/mg onmembranes which had been frozen and stored in liquid nitrogen. That¹²⁵I-[Leu³¹,Pro³⁴]PYY can bind to the rat Y5 receptor with high affinityat room temperature in isotonic buffer makes it a potentially usefulligand for characterizing the native Y5 receptor in rat tissues usingautoradiographic techniques. Care must be taken, however, to useappropriate masking agents to block potential radiolabeling of otherreceptors such as Y1 and Y4 receptors (note in Table 6 that rat Y1 andY4 bind the structural homolog [Pro³⁴]PYY). Previously published reportsof ¹²⁵I-(Leu³¹,Pro³⁴]PYY as a Y1-selective radioligand should bere-evaluated in light of new data obtained with the rat Y5 receptor(Dumont et al., 1995).

The pharmacological profile of the rat Y5 receptor was first studied byusing pancreatic polypeptide analogs in membrane binding assays. Therank order of affinity for selected compounds was derived fromcompetitive displacement of ¹²⁵I-PYY (FIG. 11). The rat Y5 receptor wascompared with cloned Y1, Y2, and Y4 receptors from human (Table 5) andrat (Table 6), all expressed transiently in COS-7 cells. One receptorsubtype absent from our panel was the Y3, human or rat, as no modelsuitable for radioligand screening has yet been identified.

TABLE 5: Pharmacological Profile of the Rat Y5 Receptor vs. Y-TypeReceptors Cloned From Human.

Binding data reflect competitive displacement of ¹²⁵I-PYY from membranesof COS-7 cells transiently expressing rat Y5 and human subtype clones.Peptides were tested at concentrations ranging from 0.001 nM to 1000 nMunless noted. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the Cheng-Prusoff equation. The data shown arerepresentative of at least two independent experiments. TABLE 5 K_(i)Values (nM) Rat Human Human Human Peptide Y5 Y4 Y1 Y2 rat/human 0.68 2.20.07 0.74 NPY porcine 0.66 1.1 0.05 0.81 NPY human 0.86 16 3.9 2.0NPY₂₋₃₆ porcine 1.2 5.6 2.4 1.2 NPY₂₋₃₆ porcine 73 38 60 2.5 NPY₁₃₋₃₆porcine >1000 304 >1000 380 NPY₂₆₋₃₆ porcine 470 120 79 3.5 C2-NPY human1.0 1.1 0.17 >130 [Leu³¹, Pro³⁴] NPY human [D- 53 >760 >1000 >1000Trp³²] NPY human NPY 480 >1000 490 >1000 free acid rat/porcine 0.64 0.140.35 1.26 PYY human PYY 0.87 0.87 0.18 0.36 human 8.4 15 41 0.70 PYY₃₋₃₆human 190 46 33 1.5 PYY₁₃₋₃₆ human 0.52 0.12 0.14 >310 [Pro³⁴] PYY humanPP 5.0 0.06 77 >1000 human PP₂₋₃₆* not 0.06 >40 >100 tested human not39 >100 >100 PP₁₃₋₃₆* tested rat PP 180 0.16 450 >1000 salmon PP 0.313.2 0.11 0.17*Tested only up to 100 nM.TABLE 6: Pharmacological Profile of the Rat Y5 Receptor vs. Y-TypeReceptors Cloned From Rat.

Binding data reflect competitive displacement of ²⁵I-PYY from membranesof COS-7 cells transiently expressing rat Y5 and rat subtype clones.Peptides were tested at concentrations ranging from 0.001 nM to 1000 nM.IC₅₀ values corresponding to 50% displacement were determined bynonlinear regression analysis and converted to K_(i) values according tothe Cheng-Prusoff equation. The data shown are representative of atleast two independent experiments. Exception: new peptides (marked witha double asterisk) were tested in one or more independent experiments.TABLE 6 K_(i) Values (nM) Peptide Rat Y5 Rat Y4 Rat Y1 Rat Y2 rat/humanNPY 0.68 1.7 0.12 1.3 porcine NPY** 0.66 1.78 0.06 1.74 frog NPY** 0.710.09 0.65 (melanostatin) human NPY₂₋₃₆ 0.86 5.0 12 2.6 porcine NPY₂₋₃₆**1.1 18 1.6 1.6 porcine NPY₃₋₃₆** 7.7 36 91 3.7 porcine NPY₁₃₋₃₆ 73 140190 31 porcine NPY₁₆₋₃₆** 260 200 140 35 porcine NPY₁₈₋₃₆** >1000 470 12porcine NPY₂₀₋₃₆** >100 360 93 porcine NPY₂₂₋₃₆** >1000 >1000 54 porcineNPY₂₆₋₃₆** >1000 >1000 >830 human 1.0 0.59 0.10 >1000 [Leu³¹, Pro³⁴] NPYporcine** 1.6 0.32 0.25 840 [Leu³¹, Pro³⁴] NPY human (O- 1.6 2.3 Methyl-Tyr²¹)NPY** human NPY free >610 >1000 720 >980 acid** porcineC2-NPY** >260 22 140 2.6 human NPY₁₋₂₄ >1000 >320 >1000 amide** human[D- 35 >630 >1000 760 Trp³²] NPY rat/porcine 0.64 0.58 0.21 0.28 PYYhuman PYY** 0.87 0.12 0.30 human PYY₃₋₃₆** 8.4 15 0.48 human PYY₁₃₋₃₆**290 130 14 human 0.52 0.19 0.25 >1000 [Pro³⁴] PYY porcine 0.64 0.240.07 >980 [Pro³⁴] PYY** avian PP** >930 >81 >320 >1000 human PP 5.0 0.0443 >1000 human PP₁₃₋₃₆** 84 >1000 >650 human PP₃₁₋₃₆** >1000 26 >10000 >10 000 human PP₃₁₋₃₆ >10,000 >100 free acid** bovine PP** 8.4 0.19120 >1000 frog PP (rana >550 >1000 720 >980 temporaria)** rat pp 2300.19 350 >1000 salmon PP 0.33 3.0 0.30 0.16 PYX-1** 920 PYX-2** >1000FLRF-amide** 5500 45 000 FMRF-amide** 18000 W(nor-L)RF- 8700 amide**

The rat Y5 receptor possessed a unique pharmacological profile whencompared with human and rat Y-type receptors. It displayed a preferencefor structural analogs of rat/human NPY (K_(i)=0.68 nM) and rat/porcinePYY (K_(i)=0.64 nM) over most PP derivatives. The high affinity forsalmon PP (K_(i)=0.31 nM) reflects the close similarity between salmonPP and rat NPY, sharing 81% of their amino acid sequence and maintainingidentity at key positions: Tyr¹, Gln³⁴, and Tyr³⁶. Both N- andC-terminal peptide domains are apparently important for receptorrecognition. The N-terminal tyrosine of NPY or PYY could be deletedwithout an appreciable loss in binding affinity (K_(i)=0.86 nM forrat/human NPY₂₋₃₆), but further N-terminal deletion was disruptive(K_(i)=73 nM for porcine NPY₁₃₋₃₆). This pattern places the bindingprofile of the Y5 receptor somewhere between that of the Y2 receptor(which receptor can withstand extreme N-terminal deletion) and that ofthe Y1 receptor (which receptor is sensitive to even a single-residueN-terminal deletion). Note that the human Y4 receptor can be describedsimilarly (K_(i)=0.06 nM for human PP, 0.06 nM for human PP₂₋₃₆, and 39nM for human PP₁₃₋₃₆) The Y5 receptor resembled both Y1 and Y4 receptorsin its tolerance for ligands containing Pro³⁴ (as in human[Leu³¹,Pro^(34])NPY, human [Pro³⁴]-PYY, and human PP). Interestingly,the rat Y5 receptor displayed a preference for human PP (K_(i)=5.0 nM)over rat PP (K_(i)=180 nM). This pattern distinguishes the rat Y5 fromthe rat Y4 receptor, which binds both human and rat PP with K_(i) values<0.2 nM. Hydrolysis of the carboxy terminal amide to free carboxylicacid, as in NPY free acid, was disruptive for binding affinity for therat Y5 receptor (K_(i)=480 nM). The terminal amide appears to be acommon structural requirement for pancreatic polypeptide family/receptorinteractions.

Several peptides shown previously to stimulate feeding behavior in ratsbound to the rat Y5 receptor with K_(i)≦5.0 nM. These include rat/humanNPY (K_(i)=0.68 nM), rat/porcine PYY (K_(i)=0.64 nM), rat/human NPY₂₋₃₆(K_(i)=0.86 nM), rat/human [Leu³¹,Pro³⁴]NPY (K_(i)=1.0 nM), and human PP(K_(i)=5.0 nM). Conversely, peptides which were relatively lesseffective as orexigenic agents bound weakly to CG-18. These includeporcine NPY₃₋₃₆ (K_(i)=73 nM), porcine C2-NPY (K_(i)=470 nM) and humanNPY free acid (K_(i)=480 nM). The rank order of K_(i) values are inagreement with rank orders of potency and activity for stimulation offeeding behavior when peptides are injected i.c.v. or directly into rathypothalamus (Clark et al., 1984; Stanley et al., 1985; Kalra et al.,1991; Stanley et al., 1992). The rat Y5 receptor also displayed moderatebinding affinity for [D-Trp³²]NPY (K_(i)=53 nM), the modified peptidereported to regulate NPY-induced feeding by Balasubramaniam et al.(1994). It is noteworthy that [D-Trp³²]NPY was ≧10-fold selective forCG-18 over the other cloned receptors studied, whether human or rat.These data clearly and definitively link the cloned Y5 receptor to thefeeding response. The cDNA corresponding to the human Y5 homologisolated from human hippocampus was transiently expressed in COS-7 cellsfor membrane binding studies. The binding of ¹²⁵I-PYY to the human Y5receptor (CG-19) was saturable over a radioligand concentration range of8 pM to 1.8 nM. Binding data were fit to a one-site binding model withan apparent K_(d) of 0.10 nM in the first experiment. Repeated testingyielded an apparent K_(d) of 0.18 nM (pK_(d)=9.76±0.11, n=4). A maximumreceptor density of 500 fmol/mg membrane protein was measured on freshmembranes. As determined by using peptide analogs within the pancreaticpolypeptide family, the human Y5 pharmacological profile bears astriking resemblance to the rat Y5 receptor (Tables 7 and 8).

TABLE 7: Pharmacological Profile of the Rat Y5 Receptor vs. the Human Y5Receptor, as Expressed Both Transiently in COS-7 and Stably in LM(tk−)Cells.

Binding data reflect competitive displacement of radioligand (either¹²⁵I-PYY or ¹²⁵I-PYY₃₋₃₆ as indicated) from membranes of COS-7 cellstransiently expressing the rat Y5 receptor and its human homolog or fromLM(tk−) cells stably expressing the human Y5 receptor. Peptides weretested at concentrations ranging from 0.001 nM to 1000 nM. IC₅₀ valuescorresponding to 50% displacement were determined by nonlinearregression analysis and converted to K_(i) values according to theCheng-Prusoff equation. New peptides are marked with a double asterisk.TABLE 7 K_(i) Values (nM) Rat Y5 Human Y5 (COS-7, Human Y5 (LM(tk−),Human Y5 ¹²⁵I- (COS-7, ¹²⁵I- (LM(tk−), Peptide PYY) ¹²⁵I-PYY) PYY)¹²⁵I-PYY₃₋₃₆) rat/human 0.68 0.15 0.89 0.65 NPY porcine 0.68 1.4 NPY**human 0.86 0.33 1.6 0.51 NPY₂₋₃₆ porcine 0.66 0.58 1.2 NPY₂₋₃₆** porcine73 110 39 NPY₁₃₋₃₆ porcine 260 300 180 NPY₁₆₋₃₆** porcine >1000 470 310NPY₁₈₋₃₆** porcine >1000 >1000 NPY₂₂₋₃₆** porcine >1000 >1000 NPY₂₆₋₃₆**human 1.0 0.72 3.0 [Leu³¹, Pro³⁴] NPY human 2.4 1.4 [Leu³¹, Pro³⁴] NPY**human NPY >610 >840 free acid** porcine 260 370 260 220 C2-NPY** human[D- 35 35 16 10 Trp³²] NPY rat/porcine 0.64 0.75 PYY human PYY** 0.870.44 1.3 0.43 human 8.4 17 8.1 1.6 PYY₃₋₃₆** human 0.52 0.34 1.7 1.7[Pro³⁴] PYY human PP 5.0 1.7 3.0 1.2 human PP₂₋₃₆** 2.1 human 290 720PP₁₃₋₃₆** human >10 000 >10 000 41 000 PP₃₁₋₃₆** human 2.0 [Ile³¹,Gln³⁴] PP** bovine PP** 8.4 1.6 7.9 5.0 rat PP 230 630 130 salmon PP0.33 0.27 0.63TABLE 8: Pharmacological Profile of the Human Y5 Receptor vs. Y-TypeReceptors Cloned From Human.

Binding data reflect competitive displacement of ¹²⁵I-PYY from membranesof COS-7 cells transiently expressing human Y5 other sub-type clones.Peptides were tested at concentrations ranging from 0.001 nM to 1000 nMunless noted. IC₅₀ values corresponding to 50% displacement weredetermined by nonlinear regression analysis and converted to K_(i)values according to the Cheng-Prusoff equation. The data shown arerepresentative of at least two independent experiments. TABLE 8 K_(i)Values (nM) Human Human Human Human Peptide Y5 Y4 Y1 Y2 rat/human NPY0.46 2.2 0.07 0.74 porcine NPY 0.68 1.1 0.05 0.81 human NPY₂₋₃₆ 0.75 163.9 2.0 porcine NPY₂₋₃₆ 0.58 5.6 2.4 1.2 porcine NPY₁₃₋₃₆ 110 38 60 2.5porcine NPY₂₆₋₃₆ >1000 304 >1000 380 porcine C2-NPY 370 120 79 3.5 human1.6 1.1 0.17 >130 [Leu³¹, Pro³⁴] NPY human [D- 35 >760 >1000 >1000Trp³²] NPY human NPY free >840 >1000 490 >1000 acid rat/porcine 0.580.14 0.35 1.26 PYY human PYY 0.44 0.87 0.18 0.36 human PYY₃₋₃₆ 17 15 410.70 human PYY₁₃₋₃₆ not 46 33 1.5 tested human 0.77 0.12 0.14 >310[Pro³⁴] PYY human PP 1.4 0.06 77 >1000 human PP₂₋₃₆* 2.1 0.06 >40 >100human PP₁₃₋₃₆* 720 39 >100 >100 rat PP 630 0.16 450 >1000 salmon PP 0.463.2 0.11 0.17*Tested only up to 100 nM.Binding Studies of hY5 Expressed in Insect Cells

Tests were initially performed to optimize expression of hY5 receptor.Infecting Sf9, Sf21, and High Five cells with hY5BB3 virus at amultiplicity of infection (MOI) of 5 and preparing membranes for bindinganalyses at 45 hrs. postinfection, we observed B_(max) ranges from 417to 820 fmoles/mg protein, with the highest expression being hY5BB3 inSf21 cells. Therefore, our next series of experiments used Sf21 cells.We next examined optimal multiplicity of infection (the ratio of viralparticles to cells) by testing MOI of 1, 2, 5 and 10. The B_(max) valueswere ˜1.1-1.2 pmoles/mg protein for any of the MOIs, suggesting thatincreasing the number of viral particles per cell is neither deleteriousnor advantageous. Since viral titer calculations are approximate, weused MOI=5 for future experiments. The last parameter we tested washours postinfection for protein expression, ranging from 45-96 hourspostinfection. We found that optimal expression occurred 45-73 hrs.postinfection. In summary, we have created a hY5 recombinant baculoviruswhich binds ¹²⁵I-PYY with a B_(max) of ˜1.2 pmoles/mg protein.

Human Y5 Homolog: Transient Expression in Baculovirus-Infected Sf21Insect Ovary Cells

Sf21 cells infected with a human Y5 baculovirus construct were harvestedas membrane homogenates and screened for specific binding of ¹²⁵I-PYYusing 0.08 nM radioligand. Specific binding was greatest (500 fmol/mgmembrane protein) for sample D-2/[41, derived from Sf-21 cells. Nospecific binding was observed after infection with the baculovirusplasmid alone (data not shown). If we make the assumption that thebinding affinity of porcine ¹²⁵-I-PYY for the human Y5 receptor is thesame whether the expression system is COS-7 or baculovirus/Sf-21 (0.18nM), the specific binding in sample D-2/[4] predicts an apparent B_(max)of 1600 fmol/mg membrane protein. The Y5 receptor yield in thebaculovirus/Sf21 expression system is therefore as good or better thanthat in COS-7. We conclude that the baculovirus offers an alternativetransfection technique amenable to large batch production of the humanY5 receptor.

Stable Expression Systems for Y5 Receptors: Characterization in BindingAssays

The cDNA for the rat Y5 receptor was stably transfected into 293 cellswhich were pre-screened for the absence of specific ¹²⁵I-PYY binding(data not shown). After co-transfection with the rat Y5 cDNA plus aG-418-resistance gene and selection with G-418, surviving colonies werescreened as membrane homogenates for specific binding of ¹²⁵I-PYY using0.08 nM radioligand. A selected clone (293 clone # 12) bound 65 fmol¹²⁵I-PYY /mg membrane protein and was isolated for further study infunctional assays.

The cDNA for the human Y5 receptor was stably transfected into bothNIH-3T3 and LM(tk−) cells, each of which were pre-screened for theabsence of specific ¹²⁵I-PYY binding (data not shown). Afterco-transfection with the human Y5 cDNA plus a G-418-resistance gene andselection with G-418, surviving colonies were screened as membranehomogenates for specific binding of ¹²⁵I-PYY using 0.08 nM radioligand.NIH-3T3 clone #8 bound 46 fmol 125I-PYY/mg membrane protein and LM(tk−)clone #7 bound 32 fmol ¹²⁵I-PYY/mg membrane protein. These two cloneswere isolated for further characterization in binding and cAMPfunctional assays. A third clone which bound 25 fmol/mg membraneprotein, LM(tk−) #3, was evaluated in calcium mobilization assays.

The human Y5 stably expressed in NIH-3T3 cells (clone #8) was furthercharacterized in saturation binding assays using ¹²⁵I-PYY. The bindingwas saturable over a concentration range of 0.4 pM to 1.9 nM. Bindingdata were fit to a one-site binding model with an apparent K_(d) of 0.30nM (pK_(d)=9.53, n=1) and an apparent B_(max) of 2100 fmol/mg membraneprotein using fresh membranes.

The human Y5 stably expressed in LM(tk−) cells (clone #7) was furthercharacterized in saturation binding assays using ¹²⁵I-PYY, ¹²⁵I-PYY₃₋₃₆,and ¹²⁵I-NPY. ¹²⁵I-PYY binding was saturable according to a 1-site modelover a concentration range of 0.4 pM to 1.9 nM, with an apparent K_(d)of 0.47 nM (pK_(d)=9.32±0.07, n=5) and an apparent B_(max) of up to 8pmol/mg membrane protein when membranes had been frozen and stored inliquid nitrogen. Peptide K_(i) values derived from ¹²⁵I-PYY binding tohuman Y5 receptors from LM(tk−) were comparable to those derived fromthe previously described human and rat Y5 expression systems (Table 7)¹²⁵ I-PYY₃₋₃₆ binding to the human Y5 in LM(tk−) cells was alsosaturable according to a 1-site model over a concentration range of 0.5pM to 2.09 nM, with an apparent K_(d) of 0.40 nM (pK_(d)=9.40, n=1) andan apparent B_(max) of 490 fmol/mg membrane protein when membranes hadbeen frozen and stored in liquid nitrogen. Peptide ligands appeared tobind with comparable affinity to human Y5 receptors in LM(tk−) cellswhether the radioligand used was ¹²⁵I-PYY or ¹²⁵I-PYY₃₋₃₆ (Table 7).Finally, ¹²⁵I-NPY binding to the human Y5 in LM(tk−) cells was saturableaccording to a 1-site model over a concentration range of 0.4 pM to 1.19nM, with an apparent K_(d) of 0.28 and an apparent B_(max) of 360fmol/mg membrane protein when membranes had been frozen and stored inliquid nitrogen.

Considering the saturation binding studies for the human and rat Y5receptor homologs as a whole, the data provide evidence that the Y5receptor is a target for multiple radioiodinated peptide analogs in thepancreatic polypeptide family, including ¹²⁵I-PYY, ¹²⁵I-NPY,¹²⁵I-PYY₃₋₃₆, and ¹²⁵I-[Leu³¹, Pro³⁴]PYY. The so-called Y1 andY2-selective radioligands (such as ¹²⁵I-[Leu³¹,Pro³⁴]PYY and¹²⁵I-PYY₃₋₃₆, respectively (Dumont et al., 1995)) should be used withcaution when probing native tissues for Y-type receptor expression.

Receptor/G Protein Interactions: Effects of Guanine Nucleotides

For a given G protein-coupled receptor, a portion of the receptorpopulation can typically be characterized in the high affinity ligandbinding site using discriminating agonists. The binding of GTP or anon-hydrolyzable analog to the G protein causes a conformational changein the receptor which favors a low affinity ligand binding state. Weinvestigated whether the non-hydrolyzable GTP analog, Gpp(NH)p, wouldalter the binding of ¹²⁵I-PYY to Y5 in COS-7 and LM(tk−) cells (FIG.19). ¹²⁵I-PYY binding to both human and rat Y5 receptors in COS-7 cellswas relatively insensitive to increasing concentrations of Gpp(NH)pranging from 1 nM to 100 μM. The human Y5 receptor in LM(tk−) cells,however, displayed a concentration dependent decrease in radioligandbinding (−85 fmol/mg membrane protein over the entire concentrationrange). The difference between the receptor preparations could beexplained by several factors, including 1) the types of G proteinsavailable in the host cell for supporting a high affinityreceptor-agonist complex, 2) the level of receptor reserve in the hostcell, and 3) the efficiency of receptor/G protein coupling, and 4) theintrinsic ability of the agonist (in this case, ¹²⁵I-PYY) to distinguishbetween multiple conformations of the receptor.

Functional Assay

Activation of all Y-type receptors described thus far is thought toinvolve coupling to pertussis toxin-sensitive G-proteins which areinhibitory for adenylate cyclase activity (G_(i) or G_(o)) (Wahlestedtand Reis, 1993). That the atypical Y1 receptor is linked to cyclaseinhibition was prompted by the observation that pertussis toxininhibited NPY-induced feeding in vivo (Chance et al., 1989); a moredefinitive analysis was impossible in the absence of the isolatedreceptor. Based on these prior observations, applicants investigated theability of NPY to inhibit forskolin-stimulated cAMP accumulation inhuman embryonic kidney 293 cells stably transfected with rat Y5receptors. Incubation of intact cells with 10 μM forskolin produced a10-fold increase in cAMP accumulation over a 5 minute period, asdetermined by radioimmunoassay. Simultaneous incubation with rat/humanNPY decreased the forskolin-stimulated cAMP accumulation by 67% instably transfected cells (FIG. 12), but not in untransfected cells (datanot shown). Applicants conclude that the rat Y5 receptor activationresults in decreased cAMP accumulation, very likely through inhibitionof adenylate cyclase activity. This result is consistent with theproposed signalling pathway for all Y-type receptors and for theatypical Y1 receptor in particular.

Peptides selected for their ability to stimulate feeding behavior inrats were able to activate the rat Y5 receptor with EC₅₀<10 nM (Kalra etal., 1991; Stanley et al., 1992; Balasubramaniam et al., 1994). Theseinclude rat/human NPY (EC₅₀=1.8 nM), rat/human NPY₂₋₃₆ (EC₅₀=2.0 nM),rat/human [Leu³¹,Pro³⁴]NPY (EC₅₀=0.6 nM), rat/porcine PYY (EC₅₀=4.0 nM),and rat/human [D-Trp³²]NPY (EC₅₀=7.5 nM) (Table 9). K_(i) values derivedfrom rat Y5-dependent binding of ¹²⁵I-PYY and peptide ligands (Table 5)were in close range of ECso values derived from rat Y5-dependentregulation of cAMP accumulation (Table 9). The maximal suppression ofcAMP produced by all peptides in Table 9 was between 84% and 120% ofthat produced by human NPY, except in the case of FLRFamide (42%). Ofparticular interest is the Y5-selective peptide [D-Trp³²]NPY. This is apeptide which was shown to stimulate food intake when injected into rathypothalamus, and which also attenuated NPY-induced feeding in the sameparadigm (Balasubramaniam, 1994). Applicants observed that [D-Trp³²]NPYbound weakly to other Y-type clones with K_(i)>500 nM (Tables 5 and 6)and displayed no activity in functional assays (Table 11). In strikingcontrast, (D-Trp³²]NPY bound to the rat Y5 receptor with a K_(i)=53 nMand was fully able to mimic the inhibitory effect of NPY onforskolin-stimulated cAMP accumulation with an EC₅₀ of 25 nm and anE_(max)=72%. That [D-Trp³²]NPY was able to selectively activate the Y5receptor while having no detectable activity at the other subtype clonesstrongly suggests that Y5 receptor activation is responsible for thestimulatory effect of [D-Trp³²]NPY on feeding behavior in vivo.

TABLE 9: Functional Activation of the Rat Y5 Receptor.

Functional data were derived from radioimmunoassay of cAMP accumulationin stably transfected 293 cells stimulated with 10 μM forskolin.Peptides were tested for agonist activity at concentrations ranging from0.03 pM to 0.3 μM. The maximum inhibition of cAMP accumulation (E_(max))and the concentration producing a half-maximal effect (EC₅₀) weredetermined by nonlinear regression analysis according to a 4 parameterlogistic equation. New peptides are marked with a double asterisk. TABLE9 Peptide E_(max) EC₅₀ (nM) rat/human 67% 1.8 NPY porcine NPY** 0.79rat/human 84% 2.0 NPY₂₋₃₆ porcine NPY₂₋₃₆** 1.2 porcine 21 NPY₁₃₋₃₆**rat/human 70% 0.6 [Leu³¹, Pro³⁴] NPY porcine 1.1 [Leu³¹, Pro³⁴] NPY**porcine C2- 240 NPY** rat/human 72% 9.5 [D-Trp³²] NPY rat/porcine 86%4.0 PYY human PYY** 1.5 human PYY₃₋₃₆** 4.9 human 1.8 [Pro³⁴] PYY**human PP** 1.4 bovine PP** 5.7 salmon PP** 0.92 rat PP** 130PYX-1** >300 PYX-2** >300 FLRFamide** 13 000

The ability of the human Y5 receptor to inhibit cAMP accumulation wasevaluated in NIH-3T3 and LM(tk−) cells, neither of which display anNPY-dependent regulation of [cAMP] without the Y5 construct. Intactcells stably transfected with the human Y5 receptor were analyzed asdescribed above for the rat Y5 cAMP assay. Incubation of stablytransfected NIH-3T3 cells with 10 uM forskolin generated an average21-fold increase in [cAMP] (n=2). Simultaneous incubation with human NPYdecreased the forskolin-stimulated [cAMP] with an E_(max) of 42% and anEC₅₀ of 8.5 nM (FIG. 20). The technique of suspending and then replatingthe Y5-transfected LM(tk−) cells was correlated with a robust andreliable cellular response to NPY-like peptides and was thereforeincorporated into the standard methodology for the functional evaluationof the human Y5 in LM(tk−). Incubation of stably transfected LM(tk−)cells prepared in this manner produced an average 7.4-fold increase in[cAMP] (n=87). Simultaneous incubation with human NPY decreased theforskolin-stimulated [cAMP] with an E_(max) of 72% and with an EC₅₀ of2.4 nM (FIG. 21). The human Y5 receptor supported a cellular response toNPY-like peptides in a rank order similar to that described for the ratY5 receptor (Table 6, 10). As the rat Y5 receptor is clearly linked byD-Trp32-NPY and other pharmacological tools to the NPY-dependentregulation of feeding behavior, the human Y5 receptor is predicted tofunction in a similar fashion. Both the human and receptor homologsrepresent useful models for the screening of compounds intended tomodulate feeding behavior by interfering with NPY-dependent pathways.

TABLE 10: Functional Activation of the Human Y5 Receptor in a cAMPRadioimmunoassay.

Functional data were derived from radioimmunoassay of cAMP accumulationin stably transfected LM(tk−) cells stimulated with 10 μM forskolin.Peptides were tested for agonist activity at concentrations ranging from0.03 pM to 0.3 μM. The maximum inhibition of cAMP accumulation (E_(max))and the concentration producing a half-maximal effect (EC₅₀) weredetermined by nonlinear regression analysis according to a 4 parameterlogistic equation. TABLE 10 % inhibition relative to Peptide human NPYEC₅₀ (nM) rat/human NPY 100% 2.7 porcine NPY 107% 0.99 rat/human NPY₂₋₃₆116% 2.6 porcine NPY₂₋₃₆ 85% 0.71 porcine NPY₁₃₋₃₆ 49 rat/human 3.0[Leu³¹, Pro³⁴] NPY porcine 1.3 [Leu³¹, Pro³⁴] NPY rat/human [D- 108% 26Trp³²] NPY rat/porcine PYY 109% 3.6 human PYY 111% 4.9 human PYY₃₋₃₆ 18human [Pro³⁴] PYY 108% 2.5 human PP 96% 14 human PP₂₋₃₆ 2.0 human 5.6[Ile³¹, Gln³⁴]PP bovine PP 4.0 salmon PP 96% 4.5TABLE 11: Binding and Functional Characterization of [D-Trp³²]NPY.

Binding data were generated as described in Tables 5 and 6. Functionaldata were derived from radioimmunoassay of cAMP accumulation in stablytransfected cells stimulated with 10 μM forskolin. [D-Trp³²]NPY wastested for agonist activity at concentrations ranging from 0.03 pM to0.3 μM. Alternatively, (D-Trp³²]NPY was included as a single spike (0.3μM) in the human PYY concentration curve for human Y1 and human Y2receptors, or in the human PP concentration curve for human Y4receptors, and antagonist activity was detected by the presence of arightward shift (from EC₅₀ to EC₅₀═). K_(b) values were calculatedaccording to the equation: K_(b)=[[D-Trp³²]NPY/ ( (EC50/EC₅₀′)−1). Thedata shown are representative of at least two independent experiments.TABLE 11 Function Receptor Binding EC₅₀ K_(b) Subtype Species K_(i) (nM)(nM) (nM) Activity Y1 Human >1000 None detected Y2 Human >1000 Nonedetected Y4 Human >1000 None detected Y5 Human 18 26 Not Determined Y1Rat >1000 Not Determined Y2 Rat >1000 Not Determined Y4 Rat >1000 NotDetermined Y5 Rat 53 9.50 AgonistFunctional Assay: Intracellular Calcium Mobilization

The intracellular free calcium concentration was increased in LM(tk−)cells stably transfected with the human Y5 receptor within 30 seconds ofincubation with 100 nM human NPY (Δ Ca²⁺=34 nM, FIG. 21D). UntransfectedLM(tk−) cells did not respond to human NPY (data not shown). The calciummobilization provides a second pathway through which Y5 receptoractivation can be measured. These data also serve to link with the Y5receptor with other cloned human Y-type receptors, all of which havebeen demonstrated to mobilize intracellular calcium in variousexpression systems (FIG. 21).

Localization Studies

The mRNA for the NPY Y5 receptor was widely distributed in rat brain,and appeared to be moderately abundant (Table 12 and FIG. 13). Themidline thalamus contained many neurons with silver grains over them,particularly the paraventricular thalamic nucleus, the rhomboid nucleus,and the nucleus reunions. In addition, moderately intense hybridizationsignals were observed over neurons in both the centromedial andanterodorsal thalamic nuclei. In the hypothalamus, a moderate level ofhybridization signal was seen over scattered neurons in the lateralhypothalamus, paraventricular, supraoptic, arcuate, and dorsomedialnuclei. In both the medial preoptic nucleus and suprachiasmatic nucleus,weak or moderate accumulations of silver grains were present. In thesuprachiasmatic nucleus, hybridization signal was restricted mainly tothe ventrolateral subdivision. In the paraventricular hypothalamus,positive neurons were observed primarily in the medial parvicellularsubdivision. TABLE 12 Distribution of NPY Y5 mRNA in the Rat CNS REGIONY5 mRNA Cerebral cortex +1 Thalamus paraventricular n. +3 rhomboid n. +3reunions n. +3 anterodorsal n. +2 Hypothalamus paraventricular n. +2lateral hypoth. area +2/+3 supraoptic n. +1 medial preoptic n. +2suprachiasmatic n. +1/+2 arcuate n. +2 Hippocampus dentate gyrus +1polymorph dentate gyrus +2 CA1 0 CA3 +1 Amygdala central amygd. n.,medial +2 anterior cortical amygd. n. +2 Olivary pretectal n. +3Anterior pretectal n. +3 Substantia nigra, pars compacta +2 Superiorcolliculus +2 Central gray +2 Rostral linear raphe +3 Dorsal raphe +1Inferior colliculus +1 Medial vestibular n. +2/+3 Parvicellular ret. n.,alpha +2 Gigantocellular reticular n., +2 alpha Pontine nuclei +1/+2

Moderate hybridization signals were found over most of the neurons inthe polymorphic region of the dentate gyrus in the hippocampus, whilelower levels were seen over scattered neurons in the CA3 region. In theamygdala, the central nucleus and the anterior cortical nucleuscontained neurons with moderate levels of hybridization signal. In themesencephalon, hybridization signals were observed over a number ofareas. The most intense signals were found over neurons in the anteriorand olivary pretectal nuclei, periaquaductal gray, and over the rostrallinear raphe. Moderate hybridization signals were observed over neuronsin the internal gray layer of the superior colliculus, the substantianigra, pars compacta, the dorsal raphe, and the pontine nuclei. Most ofthe neurons in the inferior colliculus exhibited a low level of signal.In the medulla and pons, few areas exhibited substantial hybridizationsignals. The medial vestibular nucleus was moderately labeled, as wasthe parvicellular reticular nucleus, pars alpha, and the gigantocellularreticular nucleus.

Little or no hybridization signal was observed on sections hybridizedwith the radiolabeled sense oligonucleotide probe. More importantly, inthe transfected COS-7 cells, the antisense probe hybridized only to thecells transfected with the rat Y5 cDNA (Table 13). These resultsindicate that the probe used to characterize the distribution of Y5 mRNAin rat brain is specific for this mRNA, and does not cross-hybridize toany of the other known NPY receptor mRNAs. TABLE 13 Hybridization ofantisense oligonucleotide probes to transfected COS-7 cells. Cells OligoMock rY1 rY2 rY4 rY5 rY1 − + − ND ND rY2 − − + − − rY4 − − − + − rY5 − −− − +Hybridization was performed as described in Methods. The NPY Y5 probehybridizes only to the cells transfected with the Y5 cDNA.ND = not done.In vivo Studies With Y5-Selective Compounds

The results reported above strongly support a role for the Y5 receptorin regulating feeding behavior. Accordingly, applicants have synthesizedand evaluated the binding and functional properties of several compoundsat the cloned human Y1, human Y2, human Y4, and human Y5 receptors.

As shown below in Table 14, applicants have discovered several compoundswhich bind selectively to the human Y5 receptor and act as Y5 receptorantagonists, as measured by their ability to block NPY-inducedinhibition of cAMP accumulation in forskolin-stimulated LM(tk−) cellsstably transfected with the cloned human Y5 receptor. The structures ofthe compounds described in Table 13 are shown in FIG. 22. Preliminaryexperiments indicate that compound 28 is a Y5 receptor antagonist.

Table 14: Evaluation of Human Y5 Receptor Antagonists

The ability of the compounds to antagonize the Y-type receptors isreported as the K_(b). The K_(b) is derived from the EC₅₀, orconcentration of half-maximal effect, in the presence (EC₅₀) or absence(EC₅₀′) of compound, according to the equation:K_(b)=[NPY]/((EC₅₀/EC₅₀′)−1). Results shown are representative of atleast three independent experiments. N.D.=Not determined. TABLE 14Binding Affinity (K_(i) (nM) vs. ¹²⁵I-PYY) Compound Human Receptor K_(b)(nM) — Y1 Y2 Y4 Y5 — 1 1660 1920 4540 38.9 183 2 1806 386 1280 17.8 9.65 3860 249 2290 1.27 2.1 6 4360 4610 32,900 47.5 93 7 2170 2870 705042.0 105 9 3240 >100,000 3720 108 479 10 1070 >100,000 5830 40.7 2.8 111180 >100,000 7130 9.66 1.5 17 5550 1000 8020 14 6.0 19 3550 955 1170011 23 20 16000 7760 20400 8.3 26 21 13000 1610 18500 9.8 16 22 172007570 27500 11 3.0 23 14500 617 21500 26 38 25 3240 851 13100 17 311 2623700 58200 19300 14 50 27 48700 5280 63100 28 4928 >100,000 >75,000 >100,000 19,000 N.D.

Several of these compounds were further tested using in vivo animalmodels of feeding behavior.

Since NPY is the strongest known stimulant of feeding behavior,experiments were performed with several compounds to evaluate the effectof the compounds descirbed above on NPY-induced feeding behavior insatiated rats.

First, 300 pmole of porcine NPY in vehicle (A.C.S.F.) was administeredby intracerebroventricular (i.c.v.) injection, along with i.p.administration of compound vehicle (10% DMSO/water), and the food intakeof NPY-stimulated animals was compared to food intake in animals treatedwith the vehicles. The 300 pmole injection of NPY was found tosignificantly induce food intake (p<0.05; Student-Newman-Keuls).

Using the 300 pmole dose of NPY found to be effective to stimulatefeeding, other animals were treated with the compounds byintraperitoneal (i.p.) administration, followed 30-60 minutes later byi.c.v. NPY administration, and measurement of subsequent food intake. Asshown in Table 15, NPY-induced food intake was significantly reduced inanimals first treated with the compounds (p<0.05; Student-Newman-Keuls).These experiments demonstrate that NPY-induced food intake issignificantly reduced by administration to animals of a compound whichis a Y5-selective antagonist.

Table 15. NPY-induced cumulative food intake in rats treated with eitherthe i.c.v. and i.p. vehicles (control), 300 pmole NPY alone (NPY), or inrats treated first with compound and then NPY (NPY+compound). Foodintake was measured 4 hours after stimulation with NPY. Food intake isreported as the mean±S.E.M. intake for a group of animals. TABLE 15 Foodintake (g) mean ± S.E.M. Compound 1 5 17 19 Compound 10 10 10 30 Dose(mg/kg i.p.) control 3.7 ± 0.6 2.4 ± 0.5 2.4 ± 0.7 2.9 ± 0.8 (vehiclesonly) NPY 7.4 ± 0.5 6.8 ± 1.0 5.8 ± 0.5 4.9 ± 0.4 NPY + compound 4.6 ±0.6 4.1 ± 0.4 3.8 ± 0.4 1.5 ± 0.6

Since food deprivation induces an increase in the hypothalamic NPYlevels, it has been postulated that food intake following a period offood deprivation is NPY-mediated. Therefore, the Y5 antagonists of Table14 were administered by intraperitoneal injection at a dose of 30 mg/kgto conscious rats following a 24h food deprivation. The human Y5receptor antagonists shown in Table 14 reduced food intake in thefood-deprived animals, as shown below in Table 16. The food intake ofanimals treated with test compound is reported as the percentage of thefood intake measured for control animals (treated with vehicle), i.e.,25% means the animals treated with the compound consumed only 25% asmuch food as the control animals. Measurements were performed two hoursafter administration of the test compound. TABLE 16 Two-hour food intakeof food-deprived rats. Mean Compound (%) 1 34 2 42 5 87 6 38 7 47 9 4010 74 11 15 17 27 19 36 20 35 21 80 22 55 23 58 25 32 26 73 27 84 28 NDFood intake is expressed as the percentage of intake compared to controlrats.N.D. = Not done.

These experiments indicate that the compounds of the present inventioninhibit food intake in rats, especially when administered in a range ofabout 0.01 to about 100 mg/kg rat, by either oral, intraperitoneal orintravenous administration. The animals appeared normal during theseexperiments, and no ill effects on the animals were observed after thetermination of the feeding experiments.

The binding properties of the compounds were also evaluated with respectto other cloned human G-protein coupled receptors. As shown in Table 17,below, the Y5-selective compounds described hereinabove exhibited loweraffinity for receptors other than the Y-type receptors. TABLE 17Cross-reactivity of compounds at other cloned human receptors Com-Receptor (pKi) pound α_(1d) α_(1b) α_(1a) α_(2a) α_(2b) α_(2c) H1 H2 D31 6.25 6.23 6.15 6.28 6.01 6.34 5.59 6.32 5.69 2 N.D. N.D. N.D. N.D.N.D. N.D. N.D. N.D. N.D. 5 7.24 7.36 7.63 7.39 7.29 7.63 6.65 6.68 7.246 5.68 5.73 6.54 7.14 5.79 6.35 N.D. N.D. N.D. 7 6.46 6.08 6.06 7.166.09 6.85 N.D. N.D. N.D. 9 6.45 6.26 6.57 7.04 5.00 6.81 N.D. N.D. N.D.10 6.12 5.82 6.27 8.94 5.62 6.18 N.D. N.D. N.D. 11 7.03 5.6  6.05 7.385.60 6.00 N.D. N.D. N.D. 17 6.68 7.17 7.08 6.52 6.51 7.07 6.33 5.92 6.6119 6.90 7.35 7.47 6.74 6.58 7.07 7.04 6.29 6.69 20 7.01 7.22 7.72 7.316.96 7.39 6.73 5.85 6.35 21 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.22 6.80 6.98 7.34 7.05 6.43 7.15 6.22 5.72 6.29 23 N.D. N.D. N.D. N.D.N.D. N.D. N.D. N.D. N.D. 25 6.66 6.67 7.07 6.21 5.95 6.79 6.43 6.43 5.9326 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 27 N.D. N.D. N.D. N.D.N.D. N.D. N.D. N.D. N.D. Com- Receptor (pKi) pound 5HT_(1a) 5HT₂ 5HT₇5HT_(1F) 5HT_(1E) 5HT_(1Dβ) 5HT_(1Dα) 1 4.51 6.34 6.20 5.30 5.30 5.305.42 2 N.D. N.D. N.D. N.D. N.D. N.D. N.D 5 6.33 6.41 6.00 5.30 5.30 5.555.37 6 N.D. N.D. 6.00 5.30 5.30 5.30 5.30 7 N.D. N.D. 6.64 5.30 5.305.30 5.85 9 N.D. N.D. 6.48 5.30 5.30 5.30 5.30 10 N.D. N.D. 5.87 5.305.30 5.30 5.30 11 N.D. N.D. 6.20 5.30 5.30 5.30 5.30 17 5.88 6.74 6.505.30 5.30 5.30 5.32 19 5.54 6.55 6.42 5.30 5.30 5.30 6.04 20 6.73 5.936.37 5.30 5.30 5.37 5.94 21 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 22 6.565.99 6.39 5.30 5.30 5.41 5.98 23 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 255.82 5.99 5.35 5.30 5.30 5.39 5.62 26 N.D. N.D. N.D. N.D. N.D. N.D. N.D.27 N.D. N.D. N.D. N.D. N.D. N.D. N.D.

EXPERIMENTAL DISCUSSION

In order to isolate new NPY receptor subtypes applicants choose anexpression cloning approach where a functional receptor is actuallydetected with exquisite sensitivity on the surface of transfected cells,using a highly specific iodinated ligand. Using this strategy,applicants have identified a rat hypothalamic cDNA encoding a novelY-type receptor (Y5). The fact that applicants had to screen 3.5×10⁶independent clones with a 2.7 kb average insert size to find two clonesreveals either a very strong bias against Y5 cDNA cloning in the cDNAlibrary construction procedure or that the Y5 mRNA is expressed at verylow levels in rat hypothalamic tissue. The longest reading frame in therat Y5 cDNA (CG-18) encodes a 456 amino acid protein with an estimatedmolecular weight of 50.1 kD. Given there are two N-linked glycosylationsite in the amino terminus, the apparent molecular weight could beslightly higher. Applicants have isolated the human Y5 homolog from ahuman hippocampal cDNA library. The longest reading frame in the humanY5 cDNA (CG-19) encodes a 455 amino acid protein with an estimatedmolecular weight of 50 kD. The human Y5 receptor is one amino acidshorter than the rat Y5 and shows significant amino acid differencesboth in the N-terminal and the middle of the third intracellular loopportions of the protein. The seven transmembrane domains and theextracellular loops, however, are virtually identical and the proteinmotifs found in both species homologs are identical. Both human and ratY5 receptors carry a large number of potential phosphorylation sites intheir second and third intra-cellular loops which could be involved inthe regulation of their functional characteristics.

The rat and human Y5 receptors both carry a leucine. zipper in the firstputative transmembrane domain. In such a structure, it has been proposedthat segments containing periodic arrays of leucine residues exist in analpha-helical conformation. The leucine side chains extending from onealpha-helix interact with those from a similar alpha helix of a secondpolypeptide, facilitating dimerization by the formation of a coiled coil(O'Shea et al, 1989). Usually, such patterns are associated with nuclearDNA binding protein like c-myc, c-fos and c-jun, but it is possible thatin some proteins the leucine repeat simply facilitates dimerization andhas little to do with positioning a DNA-binding region. Further evidencesupporting the idea that dimerization of specific seven transmembranereceptors can occur comes from coexpression studies withmuscarinic/adrenergic receptors where intermolecular “cross-talk”between chimeric G-protein coupled receptors has been described (Maggioet al., 1993). The tyrosine phosphorylation site found in the middle ofthis leucine zipper in transmembrane domain one (TM I) could be involvedin regulating dimerization of the Y5 receptor. The physiologicalsignificance of G-protein coupled receptor dimerization remains to beelucidated but by analogy with peptide hormone receptorsoligomerization, it could be involved in receptor activation and signaltransduction (Wells, 1994).

The nucleotide and amino acid sequence analysis of Y5 (rat and human)reveals low identity levels with all 7 TM receptors including the Y1, Y2and Y4 receptors, even in the transmembrane domains which are usuallyhighly conserved within receptor subfamilies. Applicants have namedCG-18 and CG-19 “Y5” receptors because of their unique amino acidsequence (87.2% identical with each other, ≦42% identical with the TMregions of previously cloned “Y” receptor subtypes) and pharmacologicalprofile. The name is not biased toward any one member of the pancreaticpolypeptide family. The “Y” has its roots in the original classificationof Y1 and Y2 receptor subtypes (Wahlestedt et al., 1987). The letterreflects the conservation in pancreatic polypeptide family members ofthe C-terminal tyrosine, described as “Y” in the single letter aminoacid code. The number is the next available in the Y-type series,position number three having been reserved for the pharmacologicallydefined Y3 receptor. Applicants note that the cloned human Y1 receptorwas introduced by Larhammar and co-workers as a “human neuropeptideY/peptide YY receptor of the Y1 type” (Larhammar et al., 1992).Similarly, the novel clones described herein can be described as rat andhuman neuropeptide Y/peptide YY receptors of the Y5 type.

The rat hypothalamic Y5 receptor displays a very similar pharmacologicalprofile to the pharmacologically described “atypical” Y1 receptorthought to mediate NPY-induced food intake in rat hypothalamus. Both theY5 receptor and the “feeding receptor” display a preference for NPY andPYY-like analogs, a sensitivity to N-terminal peptide deletion, and atolerance for Pro³⁴. Each would be considered Y1-like except for theanomalous ability of NPY₂₋₃₆ to bind and activate as well as NPY. Eachappears to be sensitive to changes in the mid-region of the peptideligand. For example, a study by Kalra and colleagues (1991) indicatedthat replacement of the NPY midregion by an amino-octanoic chain toproduce NPY₁₋₄-Aca-₂₅₋₃₆ dramatically reduced activity in a feedingbehavioral assay. Likewise, applicants note that the robust differencein human PP binding (K_(i)=5.0 nM) and rat PP binding (K_(i)=230) to therat Y5 receptor can be attributed to a series of 8 amino acid changesbetween residues 6-30 in the peptide ligands, with human PP bearing thecloser resemblance to human NPY. Note also that FLRFamide, a structuralanalog of the FMRFamide peptide which is reported to stimulate feedingin rats, was able to bind and activate the rat Y5 receptor albeit atrelatively high concentrations (Orosco, et al., 1989). These matchingprofiles, combined with a selective activation of the rat Y5 by thereported feeding “modulator” [D-Trp³²]NPY, support the identity of therat Y5 as the “feeding receptor” first proposed to explain NPY-inducedfeeding in rat hypothalamus. That the human Y5 receptor has apharmacological profile like that of the rat Y5 in both binding andfunctional assays suggests that the two receptors may have similarfunctions in vivo.

The distribution of Y5 mRNA in rat brain further extends the argumentfor a role of Y5 receptors in feeding behavior. The anatomical locus ofthe feeding response, for example, has been suggested to reside at leastin part in the paraventricular hypothalamic nucleus (PVN) and also inthe lateral hypothalamus, two places where Y5 mRNA was detected inabundance. Post-synaptic localization of the Y5 receptor in both ofthese regions can regulate the response to endogenously released NPY invivo. The paraventricular nucleus receives projections fromNPY-containing neurons in the arcuate nucleus, another region where Y5mRNA was detected. This indicates a pre-synaptic role for the Y5receptor in the control of NPY release via the arcuato-paraventricularprojection, and consequently in the control of feeding behavior. Thelocalization of the Y5 mRNA in the midline thalamic nuclei is alsoimportant. The paraventricular thalamic nucleus/centromedial nucleuscomplex projects heavily to the paraventricular hypothalamus and to theamygdala. As such, the Y5 receptor is a substrate for the emotionalaspect of appetitive behaviors.

Y5 receptors are highly attractive targets for appetite and weightcontrol based on several lines of research (Sahu and Kalra, 1993). NPYis the most potent stimulant of feeding behavior yet described (Clark etal., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Directinjection of NPY into the hypothalamus of rats can increase food intake˜10-fold over a 4-hour period (Stanley et al., 1992). NPY-stimulatedrats display a preference for carbohydrates over protein and fat(Stanley et al., 1985). Interestingly, NPY and NPY mRNA are increased infood-deprived rats (Brady et al., 1990; O'Shea and Gundlach, 1991) andalso in rats which are genetically obese (Sanacora et al., 1990) or madediabetic by treatment with streptozotocin (White et al., 1990). Onepotential explanation is that NPY, a potent stimulant of feedingbehavior in normal rats, is disregulated in the overweight or diabeticanimal so that food intake is increased, accompanied by obesity. Thephysiological stress of obesity increases the risk for health problemssuch as cardiovascular malfunction, osteoarthritis, andhyperinsulinemia, together with a worsened prognosis for adult-onsetdiabetes. A nonpeptide antagonist targeted to the Y5 receptor couldtherefore be effective as a way to control not only appetite and bodyweight but an entire range of obesity- and diabetes-related disorders(Dryden et al., 1994). There is also neurochemical evidence to suggestthat NPY-mediated functions are disregulated in eating disorders such asbulimia and anorexia nervosa, so that they too could be responsive totreatment by a Y5-selective drug. It has been proposed, for example,that food intake in NPY-stimulated rats mimics the massive foodconsumption associated with binge eating in bulimia (Stanley, 1993) CSFlevels of PYY but not NPY were elevated in bulimic patients whoabstained from binging, and then diminished when binging was allowed(Berrettini et al., 1988). Conversely, NPY levels were elevated inunderweight anorectic patients and then diminished as body weight wasnormalized (Kaye et al., 1990).

As described above, the human and rat in vitro expression models wereused in combination to screen for compounds intended to modulateNPY-dependent feeding behavior. Using this approach, applicants havediscovered several compounds which inhibit feeding behavior in animalmodels, which should lead to additional drug discoveries.

The Y5 pharmacological profile further offers a new standard by which toreview the molecular basis of all NPY-dependent processes; examples arelisted in Table 18. Such an exercise suggests that the Y5 receptor islikely to have a physiological significance beyond feeding behavior. Ithas been reported, for example, that a Y-type receptor can regulateluteinizing hormone releasing hormone (LHRH) release from the medianeminence of steroid-primed rats in vitro with an atypical Y1pharmacological profile. NPY, NPY₂₋₃₆, and LP-NPY were all effective at1 uM but deletion of as few as four amino acids from the N-terminus ofNPY destroyed biological activity. The Y5 may therefore represent atherapeutic target for sexual or reproductive disorders. Preliminary insitu hybridization of rat Y5 mRNA in hippocampus and elsewhere furthersuggest that additional roles will be uncovered, for example, in theregulation of memory. It is worth while considering that the Y5 is sosimilar in pharmacological profile to the other Y-type receptors that itmay have been overlooked among a mixed population of Y1, Y2 and Y4receptors. Certain functions now associated with these subtypes couldtherefore be reassigned to Y5 as our pharmacological tools grow moresophisticated (Table 18). By offering new insight into NPY receptorpharmacology, the Y5 thereby provides a greater clarity and focus in thefield of drug design. TABLE 18 Pathophysiological Conditions AssociatedWith NPY The following pathological conditions have been linked toeither 1) application of exogenous NPY, or 2) changes in levels ofendogenous NPY. 1 obesity Sahu and Kalra, 1993 2 eating disordersStanley, 1993 (anorexia and bulimia nervosa) 3 sexual/reproductiveClark, 1994 function 4 depression Heilig and Weiderlov, 1990 5 anxietyWahlestedt et al., 1993 6 cocaine Wahlestedt et al., 1991 addiction 7gastric ulcer Penner et al., 1993 8 memory loss Morley and Flood, 1990 9pain Hua et al., 1991 10 epileptic seizure Rizzi et al., 1993 11hypertension Zukowska-Grojec et al., 1993 12 subarachnoid Abel et al.,1988 hemorrhage 13 shock Hauser et al., 1993 14 circadian rhythm Albersand Ferris, 1984 15 nasal congestion Lacroix et al., 1988 16 diarrheaCox and Cuthbert, 1990 17 neurogenic Zoubek et al., 1993 voidingdysfunction

A successful strategy for the design of a Y5-receptor based drug or forany drug targeted to single G protein-coupled receptor subtype involvesthe screening of candidate compounds 1) in radioligand binding assays soas to detect affinity for cross-reactive G protein-coupled receptors,and 2) in physiological assays so as to detect undesirable side effects.In the specific process of screening for a Y5-selective drug, thereceptor subtypes most likely to cross-react and therefore mostimportant for radioligand binding screens include the other “Y-type”receptors, Y1, Y2, Y3, and Y4. Cross-reactivity between the Y5 and anyof the other subtypes could result in potential complications assuggested by the pathophysiological indications listed in Table 18. Indesigning a Y5 antagonist for obesity and appetite control, for example,it is important not to design a Y1 antagonist resulting in hypertensionor increased anxiety, a Y2 antagonist resulting in memory loss, or a Y4antagonist resulting in increased appetite. TABLE 19 Y-Type ReceptorIndications Y-type Receptor Receptor Drug Indications Subtype ActivityReference obesity, atypical Y1 antagonist Sahu and appetite Kalra,disorder 1993 adult onset atypical Y1 antagonist Sahu and diabetesKalra, 1993 bulimia atypical Y1 antagonist Stanley, nervosa 1993pheochromocytoma- Y1 antagonist Grouzman induced et al., hypertension1989 subarachnoid Y1 antagonist Abel et hemorrhage al., 1988 neurogenicY1 antagonist Zukowska- vascular Y2 antagonist Grojec et hypertrophyal., 1993 epileptic Y2 antagonist Rizzi et seizure al., 1993hypertension: peripheral Y1 antagonist Grundemar central, central Y3agonist and peripheral central Y2 antagonist Hakanson, regulation 1993Barraco et al., 1991 obesity, Y4 or PP agonist Malaisse- appetite Lagaeet disorder al., 1977 anorexia atypical Y1 agonist Berrettin nervosa iet al., 1988 anxiety Y1 agonist Wahlested t et al., 1993 cocaine Y1agonist Wahlested addiction t et al., 1991 stress- Y1 agonist Penner etinduced Y4 or PP agonist al., 1993 gastric ulcer memory loss Y2 agonistMorley and Flood, 1990 pain Y2 agonist Hua et al., 1991 shock Y1 agonistHauser et al., 1993 sleep Y2 not clear Albers disturbances, and jet lagFerris, 1984 nasal Y1 agonist Lacroix decongestion Y2 agonist et al.,1988 diarrhea Y2 agonist Cox and Cuthbert, 1990

The cloning of the Y5 receptor from human and rat is especially valuablefor receptor characterization based on in situ localization, anti-sensefunctional knock-out, and gene induction. These studies will generateimportant information related to Y5 receptor function and itstherapeutic significance. The cloned Y5 receptor lends itself tomutagenesis studies in which receptor/ligand interactions can bemodeled. The Y5 receptor further allows us to investigate thepossibility of other Y-type receptors through homology cloning. Thesecould include new receptor subtypes as well as Y5 species homologs forthe establishment of experimental animal models with relevance for humanpathology. The Y5 receptor therefore represents an enormous opportunityfor the development of novel and selective drug therapies, particularlythose targeted to appetite and weight control, but also for memory loss,depression, anxiety, gastric ulcer, epileptic seizure, pain,hypertension, subarachnoid hemorrhage, sleeping disturbances, nasalcongestion, neurogenic voiding dysfuncion, and diarrhea.

In particular, the discovery of Y5-selective antagonists which inhibitfood intake in rats provides a method of modifying feeding behavior in awide variety of vetebrate animals.

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1-42. (canceled)
 43. An isolated nucleic acid encoding a Y5 receptor.44-59. (canceled)
 60. A purified Y5 receptor protein. 61-92. (canceled)93. A nucleic acid probe comprising a nucleic acid of at least 15nucleotides capable of specifically hybridizing with a unique sequenceincluded within the sequence of a nucleic acid encoding a Y5 receptor ofclaim
 43. 94. A nucleic acid probe of claim 93, wherein the nucleic acidis DNA.
 95. A nucleic acid probe of claim 93, wherein the nucleic acidis RNA. 96-98. (canceled)
 99. An antibody capable of binding to a Y5receptor of claim
 43. 100. An antibody of claim 99, wherein the Y5receptor is a human Y5 receptor.
 101. An antibody capable ofcompetitively inhibiting the binding of the antibody of claim 99 to a Y5receptor.
 102. An antibody of claim 99 wherein the antibody is amonoclonal antibody.
 103. A monoclonal antibody of claim 102 directed toan epitope of a Y5 receptor present on the surface of a Y5 receptorexpressing cell. 104-108. (canceled)
 109. A pharmaceutical compositionwhich comprises an amount of the antibody of claim 99 effective to blockbinding of a ligand to the Y5 receptor and a pharmaceutically acceptablecarrier. 110-147. (canceled)
 148. A method of screening a plurality ofchemical compounds not known to inhibit the activation of a Y5 receptorto identify a compound which inhibits the activation of the Y5 receptor,which comprises (a) contacting a cell transfected with and expressingthe Y5 receptor with the plurality of compounds in the presence of aknown Y5 receptor agonist, under conditions permitting activation of theY5 receptor; (b) determining whether the activation of the Y5 receptoris reduced in the presence of the plurality of compounds, relative tothe activation of the Y5 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the Y5 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the Y5 receptor. 149-160. (canceled)
 161. Amethod of detecting the presence of a human Y5 receptor on the surfaceof a cell which comprises contacting the cell with the antibody of claim99 under conditions permitting binding of the antibody to the receptor,detecting the presence of the antibody bound to the cell, and therebydetecting the presence of a human Y5 receptor on the surface of thecell. 162-173. (canceled)
 174. A method of preparing the purified Y5receptor of claim 60 which comprises: (a) inducing cells to express Y5receptor; (b) recovering the receptor from the induced cells; and (c)purifying the receptor so recovered.
 175. A method of preparing thepurified Y5 receptor of claim 60 which comprises: (a) inserting nucleicacid encoding Y5 receptor in a suitable vector; (b) introducing theresulting vector in a suitable host cell; (c) placing the resulting cellin suitable condition permitting the production of the isolated Y5receptor; (d) recovering the receptor produced by the resulting cell;and (e) purifying the receptor so recovered.